Anti-KSA/IL-2 fusion proteins with reduced immunogenicity

ABSTRACT

The invention relates to artificial modified proteins, preferably fusion proteins, having a reduced immunogenicity compared to the parent non-modified molecule when exposed to a species in vivo. The invention relates, above all, to novel immunoglobulin fusion proteins which essentially consist of an immunoglobulin molecule or a fragment thereof covalently fused via its C-terminus to the N-terminus of a biologically active non-immunoglobulin molecule, preferably a polypeptide or protein or a biologically active fragment thereof. In a specific embodiment, the invention relates to fusion proteins consisting of an Fc portion of an antibody which is fused as mentioned to the non-immunological target molecule which elicits biological or pharmacological efficacy. The molecules of the invention have amino acid sequences which are altered in one or more amino acid residue positions but have in principal the same biological activity as compared with the non-altered molecules. The changes are made in regions of the molecules which are identified as T-cell epitopes, which contribute to an immune reaction in a living host. Thus, the invention also relates to a novel method of making such fusion proteins by identifying said epitopes comprising calculation of T-cell epitope values for MHC Class II molecule binding sites in a peptide by computer-aided methods.

This application is the National Stage of International Application No.PCT/EP02/01690, filed on Feb. 18, 2002, which claims priority fromEuropean Patent Application No. 01108291.4, filed on Apr. 5, 2001 andEuropean Patent Application No. 01103955.9, filed on Feb. 19, 2001.

TECHNICAL FIELD OF THE INVENTION

The invention relates to modified artificial proteins, preferably fusionproteins, having a reduced immunogenicity compared to the parentnon-modified molecule when exposed to a species in vivo. Especially, theinvention relates to proteins that are as single component normally notstrongly immunogenic, but which have enhanced immunogenicity whenattached to a second protein moiety to form an, as a rule, artificialfusion protein. The invention relates, above all, to modified and, thus,novel immunoglobulin (Ig) fusion proteins which essentially consist ofan immunoglobulin molecule or a fragment thereof covalently fused viaits C-terminus to the N-terminus of a biologically activenon-immunoglobulin molecule, preferably a polypeptide or protein or abiologically active fragment thereof. In a specific embodiment, theinvention relates to fusion proteins consisting of an Fc portion of anantibody which is fused as mentioned to the non-immunological targetmolecule which elicits biological or pharmacological efficacy.

The molecules of the invention have amino acid sequences which arealtered in one or more amino acid residue positions but have inprincipal the same biological activity as compared with the non-alteredmolecules. The changes are made in regions of the molecules which areidentified as T-cell epitopes, which contribute to an immune reaction ina living host. Thus, the invention also relates to a novel method forpreparing said fusion proteins by identifying said epitopes comprisingcalculation of T-cell epitope values for MHC Class II molecule bindingsites in a peptide by computer-aided methods.

BACKGROUND OF THE INVENTION

Therapeutic fusion proteins are, as a rule, artificial molecules, whichare produced to combine known favorable properties of the singlecomponents or to create new properties. For example, a fusion proteinmay contain an immunogenic moiety that causes a normally non-immunogenicfusion partner to become immunogenic. In other cases, each of thecomponents are immunogenic and the fusion molecule has kept this usuallyundesired property. Finally, it is possible that fusing non or lessimmunogenic components the fusion product is immunogenic by creating thebonds, especially the junction region.

Fusion proteins of specific interests in this context areimmunoconjugates. Immunoconjugates are known since a couple of years andmany of them have shown pharmacological efficacy in vitro and in vivo.Immunoconjugates are chimeric molecules consisting, as a rule, of aportion deriving from an immunoglobulin or a fragment thereof and atarget polypeptide or protein which is linked to the immunoglobulinmolecule. Originally, immunoconjugates were prepared consisting of acomplete antibody and a cytotoxic agent like a cytokine which was fusedvia its N-terminus to the C-terminus of the constant domain of theimmunoglobulin, or alternatively, via its C-terminus to the N-terminusof the variable region of the antibody (see, for example EP 0439 095, WO92/08495, U.S. Pat. No. 5,349,053, EP 0659 439, EP 0706 799). Thesechimeric molecules are bi-functional by targeting a specific antigen,for example, on a tumor cell surface by means of the binding siteswithin the CDRs of the variable domain of the antibody portion or afragment thereof, and by the simultaneous cytotoxic effect of thecytokine which is coupled to the immunoglobulin and thus can,theoretically, only or predominantly attack the targeted cell. In thiscontext, also tri- and multi-functional immunoconjugates were developedincluding constructs consisting of sFv-, Fab-, Fab′ or F(ab′)2 fragmentsof different antibodies, wherein in each case the targeting function ofthe immunoglobulin portion was advantageously used.

Another immunoglobulin modification is the use of the Fc region ofantibodies. Antibodies comprise two functionally independent parts, avariable domain known as “Fab”, “Fab”, “F(ab′)2”, dependent on the kindof digestion of the molecule, which bind antigen, and a constant domain,known as “Fc” which provides the link to effector functions such ascomplement or phagocytic cells. The Fc portion of an immunoglobulin hasa long plasma half-life, whereas the Fab fragments are short-lived(Capon, et al., Nature 337: 525–531 (1989)).

Therapeutic protein products have been constructed using the Fc domainto provide longer half-life or to incorporate functions such as Fcreceptor binding, protein A binding, complement fixation and placentaltransfer which all reside in the Fc proteins of immunoglobulins. Forexample, the Fc region of an IgG1 antibody has been fused to theN-terminal end of CD30-L, a molecule which binds CD30 receptorsexpressed on Hodgkin's Disease tumor cells, anaplastic lymphoma cells,T-cell leukemia cells and other malignant cell types (U.S. Pat. No.5,480,981). IL-10, an anti-inflammatory and antirejection agent has beenfused to murine Fcγ2a in order to increase the cytokine's shortcirculating half-life (Zheng et al., The Journal of Immunology, 154:5590–5600 (1995)). Studies have also evaluated the use of tumor necrosisfactor receptor linked with the Fc protein of human IgG1 to treatpatients with septic shock (Fisher et al., N. Engl. J. Med., 334:1697–1702 (1996); Van Zee et al., The Journal of Immunology, 156:2221–2230 (1996)). Fc has also been fused with CD4 receptor to produce atherapeutic protein for treatment of AIDS (see: Capon et al., Nature,337:525–531 (1989)). Principally, Fc can be fused to the target proteinor peptide via its C- or N-terminus using the N- and C-terminus of theprotein, respectively. A chimer of Fc and TNF and EPO was disclosed inEP 0464 533 (Hoechst/General Hospital), wherein the N-terminus of Fc wascoupled to the C-terminus of the protein (X-Fc). The identicalconjunction was selected for leptin-Fc chimers disclosed in WO 97/00319(SKB) and WO 97/24440 (Genentech). There are a lot of publications andpatent applications describing the opposite linkage of Fc-proteinchimers (Fc-X), such as Fc-(IL-2), Fc-EPO, Fc-PSMA, Fc-(IL-12), Fc-TNFa,Fc-(GM-CSF), Fc-TNFR, Fc-endostatin, Fc, angiostatin, Fc-gp120,Fc-leptin, Fc-IFNa, Fc-(G-CSF). Examples are WO 96/08570, (Fuji/MerckKGaA), WO 98/28427 (Amgen), WO 99/02709 (Beth Israel Medical CareCenter) and WO 99/58662 (Fuji/Merck KGaA). WO 00/24782 (Amgen) disclosesa huge number of possible Fc-X conjugates, wherein the linkage betweenthe two partners may be Fc-X or X-Fc. An extensive development of Fc-Xmolecules was realized by Lexigen/Merck KgaA as disclosed in U.S. Pat.No. 5,541,087, WO 99/43713, WO 99/29732, WO 99/52562, WO 99/53958, WO00/11033, WO 01/07081, PCT/EP00/10843. Thus, X-Fc and Fc-X moleculeswhich have “lost” their antigen binding sites, as well as molecules,wherein the binding sites and thus their antigen-specific targetingfunctions are conserved, are of great interest as promising therapeuticproteins and there exists a further need to develop analoguecompositions for different clinical application. Non-natural therapeuticproteins are often particularly immunogenic. For example, Enbrel is afusion protein consisting of an extracellular domain of a Tumor NecrosisFactor Receptor (TNF-R) fused to an Fc region of an antibody. About 16%of patients treated with Enbrel have been reported to develop antibodiesto this fusion protein (Physician's Desk Reference [2001] p. 3372).Similarly, a fusion protein consisting of erythropoietin (Epo) andgranulocyte/macrophage-colony stimulating factor (GMCSF) was found to behighly immunogenic (Coscarella A, et al. [1998] Cytokine 10:964–9;Coscarella A, Mol Biotechnol. [1998] 10:115–22). When injected into aprimate, Epo-GMCSF fusion proteins were found to induce a strongantibody response to the Epo moiety of the fusion protein, resulting inanemia. Ceredase™ and Cerezyme™ are forms of the lysosomal enzymeglucocerebrosidase used to treat Gaucher's disease; as a result ofgenetic engineering, glucocerebrosidase is attached to an unusualhigh-mannose glycosylation. Patients with Gaucher's disease lackglucocerebrosidase in their lysosomes, and as a result the patients'macrophages tend to accumulate lipids and become foam cells (TheMetabolic and Molecular Bases of Inherited Disease, 8^(th) Edition[2001] Scriver et al. eds. Chapter 146, “Gaucher Disease.” p.3635–3668).After administration of Ceredase™ or Cerezyme™, the therapeutic proteinis bound by mannose receptors on macrophages, endocytosed, andtrafficked through the endosomes to the lysosome, which is its properlocation. Patients treated with Ceredase often develop antibodies toglucocerebrosidase (Pastores G M, et al., Blood [1993] 82:408–16;Physicians' Desk Reference [2001] p. 1325–1326). Such antibodies caninterfere with treatment (Brady R O, et al., Pediatrics. [1997]100(6):E11.). In a Phase I clinical trial using an antibody-cytokinefusion protein, several patients developed antibody responses to thetherapeutic fusion protein. In this case, the antibody moiety was ahumanized form of the 14.18 antibody, and the cytokine was interleukin-2(IL-2). Many of the reactive patients' sera included significant levelsof anti-idiotype antibodies.

Therapeutic use of a number of peptides, polypeptides and proteins iscurtailed because of their immunogenicity in mammals, especially humans.For example, when murine antibodies are administered to patients who arenot immunosuppressed, a majority of such patients exhibit an immunereaction to the introduced foreign material by making human anti-murineantibodies (HAMA) (e.g. Schroff, R. W. et at (1985) Cancer Res. 45:879–885; Shawler, D. L. et at (1985) J. Immunol. 135: 1530–1535). Thereare two serious consequences. First, the patient's anti-murine antibodymay bind and clear the therapeutic antibody or immunoconjugate before ithas a chance to bind, for example to a tumor, and perform itstherapeutic function. Second, the patient may develop an allergicsensitivity to the murine antibody and be at risk of anaphylactic shockupon any future exposure to murine immunoglobulin.

Several techniques have been employed to address the HAMA problem andthus enable the use in humans of therapeutic monoclonal antibodies (see,for example, WO89/09622, EP0239400, EP04383 10, WO91/09967). Theserecombinant DNA approaches have generally reduced the mouse geneticinformation in the final antibody construct whilst increasing the humangenetic information in the final construct. Notwithstanding, theresultant “humanized” antibodies have, in several cases, still elicitedan immune response in patients (Issacs J. D. (1990) Sem. Immunol. 2:449,456; Rebello, P. R. et al (1999) Transplantation 68: 1417–1420). Acommon aspect of these methodologies has been the introduction into thetherapeutic antibody, usually of rodent origin, of amino acid residues,even significant tracts of amino acid residue sequences, identical tothose present in human antibody proteins. For antibodies, this processis possible owing to the relatively high degree of structural (andfunctional) conservatism among antibody molecules of different species.For potentially therapeutic peptides, polypeptides and proteins,however, where no structural homologue may exist in the host species(e.g., human) for the therapeutic protein, such processes are notapplicable. Furthermore, these methods have assumed that the generalintroduction of a human amino acid residue sequence will render there-modeled antibody non-immunogenic. It is known, however, that certainshort peptide sequences (“T-cell epitopes”) can be released during thedegradation of peptides, polypeptides or proteins within cells andsubsequently be presented by molecules of the major histocompatabilitycomplex (MHC) in order to trigger the activation lof T-cells. Forpeptides presented by MHC Class II, such activation of T-cells can thengive rise to an antibody response by direct stimulation of B-cells toproduce such antibodies. Accordingly, it would be desirable to eliminatepotential T-cell epitopes from a peptide, polypeptide or a protein. Evenproteins of human origin and with the same amino acid sequences as occurwithin humans can still induce an immune response in humans. Notableexamples include therapeutic use of granulocyte-macrophage colonystimulating factor (Wadhwa, M. et al (1999) Clin. Cancer Res. 5:1353–1361) and interferon alpha 2 (Russo, D. et al (1996) Bri. J. Haem.94: 300–305; Stein, R. et al (1988) New Engl. J. Med. 318: 1409–1413).

During the last couple of years several techniques were published whichsuggest solutions for rendering antibodies and target proteins havingdifferent biological functions non- or at least less immunogenic.Examples are: WO 92/10755 and WO 96/40792 (Novo Nordisk), EP 0519 596(Merck & Co.), EP 0699 755 (Centro de Immunologia Moelcular), WO98/52976 and WO 98/59244 and WO 00/34317 (Biovation Ltd.).

The general methods disclosed in the prior art and regarding theelimination of T-cell epitopes from proteins (e.g. WO 98/52976, WO00/34317) comprise the following steps:

-   -   (a) Determining the amino acid sequence of the polypeptide or        part thereof    -   (b) Identifying one or more potential T-cell epitopes within the        amino acid sequence of the protein by any method including        determination of the binding of the peptides to MHC molecules        using in vitro or in silico techniques or biological assays.    -   c) Designing new sequence variants with one or more amino acids        within the identified potential T-cell epitopes modified in such        a way to substantially reduce or eliminate the activity of the        T-cell epitope as determined by the binding of the peptides to        MHC molecules using in vitro or in silico techniques or        biological assays. Such sequence variants are created in such a        way to avoid creation of new potential T-cell epitopes by the        sequence variations unless such new potential T-cell epitopes        are, in turn, modified in such a way to substantially reduce or        eliminate the activity of the T-cell epitope.    -   (d) Constructing such sequence variants by recombinant DNA        techniques and testing said variants in order to identify one or        more variants with desirable properties.

Other techniques exploiting soluble complexes of recombinant MHCmolecules in combination with synthetic peptides and able to bind toT-cell clones from peripheral blood samples from human or experimentalanimal subjects have been used in the art [Kern, F. et al (1998) NatureMedicine 4:975–978; Kwok, W. W. et al (2001) TRENDS in Immunology 22:583–588] and may also be exploited in an epitope identificationstrategy.

The potential T-cell epitopes are generally defined as any amino acidresidue sequence with the ability to bind to HMC Class II molecules.Such potential T-cell epitopes can be measured to establish MHC binding.In the general understanding the term “T-cell epitope” is an epitopewhich when bound to MHC molecules can be recognized by the T-cellreceptor, and which can, at least in principle, cause the activation ofthese T-cells. It is, however, usually understood that certain peptideswhich are found to bind to MHC Class II molecules may be retained in aprotein sequence because such peptides are tolerated by the immunewithin the organism into which the final protein is administered.

The invention is conceived to overcome the practical reality thatsoluble proteins introduced into an autologous host with therapeuticintent, can trigger an immune response resulting in development of hostantibodies that bind to the soluble protein. One example amongst othersis interferon alpha 2 to which a proportion of human patients makeantibodies despite the fact that this protein is produced endogenously[Russo, D. et al (1996) Brit. J. Haem. 94: 300–305; Stein, R. et al(1988) New Engl. J. Med. 318: 1409–1413]

MHC Class II molecules are a group of highly polymorphic proteins whichplay a central role in helper T-cell selection and activation. The humanleukocyte antigen group DR (HLA-DR) are the predominant isotype of thisgroup of proteins and the major focus of the present invention. However,isotypes HLA-DQ and HLA-DP perform similar functions, hence the presentinvention is equally applicable to these. MHC HLA-DR molecules arehomo-dimers where each “half” is a hetero-dimer consisting of α and βchains. Each hetero-dimer possesses a ligand binding domain which bindsto peptides varying between 9 and 20 amino acids in length, although thebinding groove can accommodate a maximum of 9–11 amino acids. The ligandbinding domain is comprised of amino acids 1 to 85 of the α chain, andamino acids 1 to 94 of the β chain. DQ molecules have recently beenshown to have an homologous structure and the DP family proteins arealso expected to be very similar. In humans approximately 70 differentallotypes of the DR isotype are known, for DQ there are 30 differentallotypes and for DP 47 different allotypes are known. Each individualbears two to four DR alleles, two DQ and two DP alleles. The structureof a number of DR molecules has been solved and such structures point toan open-ended peptide binding groove with a number of hydrophobicpockets which engage hydrophobic residues (pocket residues) of thepeptide [Brown et al Nature (1993) 364: 33; Stern et al (1994) Nature368: 215]. Polymorphism identifying the different allotypes of class IImolecule contributes to a wide diversity of different binding surfacesfor peptides within the peptide binding grove and at the populationlevel ensures maximal flexibility with regard to the ability torecognize foreign proteins and mount an immune response to pathogenicorganisms.

There is a considerable amount of polymorphism within the ligand bindingdomain with distinct “families” within different geographicalpopulations and ethnic groups. This polymorphism affects the bindingcharacteristics of the peptide binding domain, thus different “families”of DR molecules will have specificities for peptides with differentsequence properties, although there may be some overlap. Thisspecificity determines recognition of Th-cell epitopes (Class II T-cellresponse) which are ultimately responsible for driving the antibodyresponse to B-cell epitopes present on the same protein from which theTh-cell epitope is derived. Thus, the immune response to a protein in anindividual is heavily influenced by T-cell epitope recognition which isa function of the peptide binding specificity of that individual'sHLA-DR allotype. Therefore, in order to identify T-cell epitopes withina protein or peptide in the context of a global population, it isdesirable to consider the binding properties of as diverse a set ofHLA-DR allotypes as possible, thus covering as high a percentage of theworld population as possible.

A principal factor in the induction of an immune response is thepresence within the protein of peptides that can stimulate the activityof T-cell via presentation on MHC class II molecules. In order toeliminate or reduce immunogenicity, it is thus desirable to identify andremove T-cell epitopes from the protein.

According to the above-cited methods and related processes severalbiological molecules, basically usual target proteins and antibodieshave been prepared which reveal reduced immunogenicity andallergenicity. Examples are: WO 99/55369 (SKB), WO 99/40198 and WO96/21016 (Leuven Research & Development VZW), WO 00/08196 (DukeUniversity), WO 96/21036 (Chiron Viragen), WO 97/31025 (Chiron Corp.),WO 98/30706 (Alliance Pharmaceutical Corp.).

In all these applications cited above single proteins or antibodieseliciting a lower immune response were disclosed; there is no hint thatfusion proteins, above all immunoglobulin fusion proteins werecompletely or partially de-immunized, especially by reducing the numberof T-cell epitopes within the sequence of said molecules by means ofpartially computational methods. In WO 97/24137 (Tannox Biosystems Inc.)a IFNα-Fc chimer is disclosed which contains a non-immunogenic linkermolecule between the N-terminus of the Fc portion and the C-terminus ofIFNα.

Therefore, it is still a need to provide for biological molecules, suchas immunoconjugates, which are not or less immunogenic. Above all, it isof specific interest to provide for Fc-conjugates, preferably Fc-Xchimers, wherein X is a selected protein or polypeptide of therapeuticinterest.

SUMMARY OF THE INVENTION

The present invention relates to four general aspects:

-   (a) a novel application of the details of the immune response    mechanism to situations involving fusion proteins and other    artificial proteins, to help determine when an engineered or novel    protein is likely to be immunogenic and therefore when application    of a deimmunization methodology is appropriate,-   (b) novel biologically active artificial proteins to be administered    especially to humans and in particular for therapeutic use,-   (c) a method of designing improved, less immunogenic artificial    proteins that normally have enhanced immunogenicity, the method    comprising identification of one or more candidate T-cell epitopes    in the artificial protein and introducing a mutation that removes    one or more T-cell epitopes, and-   (d) a convenient and effective computational method for the    identification and calculation of T-cell epitopes for a globally    diverse number of MHC Class II molecules and, based on this    knowledge, for designing and constructing new sequence variants of    biological molecules with improved properties. Once T-cell epitopes    have been identified in an artificial protein, they are removed by    mutation as described in (c).

Artificial proteins that have a component capable of binding to asurface receptor on a cell of the immune system are, in general,particularly immunogenic. Artificial proteins that are immunogenic as aconsequence of having a moiety that binds to an immune cell surfacereceptor are particularly good candidates for the methods of theinvention for reducing immunogenicity.

Without wishing to be bound by theory, FIGS. 1–6 present diagrams ofartificial proteins containing moieties that bind to immune cell surfacereceptors such as an Fc receptor, a cytokine receptor, or anoligosaccharide receptor.

One method of the invention consists of the steps of identifyingartificial proteins that contain moieties that bind to an immune cellsurface receptor, which may be done by sequence inspection, identifyingcandidate T-cell epitopes in the artificial protein, designing mutantderivatives of the artificial protein in which the number of T-cellepitopes is reduced, producing one or more mutant derivatives, testingthe mutant derivatives for activity and optionally other desiredproperties, and choosing a mutant derivative that has an optimal balanceof reduced T-cell epitopes, retained activity, and optionally otherretained desired properties. Other desired properties may include, butare not limited to, pharmacokinetic properties and protein expressionand assembly characteristics.

Artificial proteins that tend to form aggregates are a second categoryof proteins that can be improved by the methods of the invention.

One class of artificial proteins that can particularly be improved bythe invention are Ig fusion proteins, such as fusion proteins comprisingan entire antibody, as well as Fc-X and X-Fc fusion proteins. Inparticular, immunoglobulin fusion proteins comprising a functional Fcreceptor binding site can be particularly improved by methods of theinvention.

The invention provides improved forms of such antibody fusion proteins,which include fusion proteins comprising V regions that recognizetumor-specific antigens, other tissue-specific antigens, or otherdisease-specific antigens. In one preferred embodiment, each of theseantibodies is fused to a cytokine, such as IL-2.

For example, the invention provides fusion proteins comprising thetumor-directed anti-EpCAM antibody KS 1/4 and anti-GD2 antibody 14.18,in which the V regions of the antibody contain mutations that removeT-cell epitopes.

In a distinct embodiment, the moiety that is fused to the antibodymoiety is mutated such that T-cell epitopes are removed. For example,the invention discloses an antibody-IL-2 fusion protein in which theIL-2 moiety has been altered to remove T-cell epitopes.

A second general class of Ig fusion proteins that can be significantlyimproved are the Fc-X and X-Fc fusion proteins. Without wishing to bebound by theory, it is thought that these proteins are particularlyimmunogenic because the Fc receptor binding site, which is normallysomewhat sterically blocked by the light chain in an intact antibody, isexposed. In any case, it has been empirically established that an Fcfusion protein can be more immunogenic than the fusion partner by itself(WO01/07081).

Another class of immunogenic fusion proteins are proteins that are fusedto a cytokine. Without wishing to be bound by theory, it may be thatthese proteins are particularly immunogenic because when the fusionpartner protein binds to an immune cell, for example a cell bearing anantibody that recognizes the fusion partner protein, the cytokinestimulates the cell in some way (see FIG. 4).

A class of artificial proteins that are particularly immunogenic arenormal proteins that contain an inappropriate oligosaccharide. Forexample, a protein containing an oligosaccharide that is bound by aspecific receptor on an immune cell is often found to be immunogenic.For example, a protein, preferably a protein such asbeta-glucocerebrosidase that can be used to treat a lysosomal storagedisorder, contains a high mannose oligosaccharide. Such an immunogenicprotein shows significantly reduced immunogenicity when modifiedaccording to the invention.

The invention provides less immunogenic forms of the following proteinmoieties that are incorporated into otherwise immunogenic fusionproteins such as: erythropoietin, leptin, keratinocyte growth factor,G-CSF GM-CSF, IL-IR antagonist, sTNFR, TNF inhibitor, sTNFR-Fc(Enbrel®), BNTF, CNTF, members of the interferon family, hGH,β-glucocerebrosidase. All these biologically active protein moietieslisted above derive from well known non-modified (parent) proteinmoieties according to the invention.

The modified proteins according to the invention may be produced by themethod indicated in Section “Detailed Description of the Invention”. Themethod includes a novel method for identification T-cell epitopes bycomputational means. This method step is preferred according to theinvention and described in more detail in EXAMPLE 1.

The invention discloses and claims as preferred embodiments of theinvention altered or modified fusion proteins derived from parent fusionproteins, said parent fusion protein essentially consisting of animmunoglobulin molecule or a fragment thereof and a non-immunoglobulintarget polypeptide (X), which is linked preferably by its N-terminal tothe C-terminal of the immunoglobulin molecule or a fragment thereof,wherein the altered fusion protein has an amino acid sequence differentfrom that of said parent fusion protein and exhibits reducedimmunogenicity relative to the parent fusion protein when exposed to theimmune system of a given species, that is preferably human.

The strategies that are used in practice according to the invention toreduce the immunogenicity of an immunogenic fusion protein areillustrated in detail for the antibody-cytokine fusions. These generalstrategies include:

-   -   Examining the amino acid sequences in the fusion protein and        prioritizing them with respect to likely immunogenicity, based        on the expected presence and abundance of the sequences during        negative selection of T-cells in the thymus. For example,        completely non-self epitopes are identified, and are the highest        priority for removal of T-cell epitopes by mutation. The lowest        priority for removal of T-cell epitopes are sequences that are        present in abundant serum proteins, such as antibody constant        regions or sequences that are found in un-rearranged antibody V        regions. An intermediate priority for removal of T-cell epitopes        by mutation are self sequences that are found in low abundance        proteins, such as cytokines. Without wishing to be bound by        theory, it is expected that low abundance proteins may not be        present in the thymus in high enough amounts to promote negative        T-cell selection, and may thus be recognized as non-self T-cell        epitopes.    -   When a region is chosen for removal of T-cell epitopes by        mutation, it is compared with naturally occurring human        sequences found in abundant proteins. Mutations are introduced        to make any non-self sequences more similar to self sequences.        For example, to reduce the immunogenicity of a mouse V region,        the sequence is compared to un-rearranged human V regions and        the most closely related sequence is found. “Veneering” changes        are introduced, in which some amino acids are converted from        mouse to human. This has the effect of converting some non-self        T-cell epitopes into self T-cell epitopes, a method for reducing        immunogenicity disclosed by U.S. Pat. No. 5,712,120, and also        has the effect of removing some B cell epitopes. However, it is        still necessary to remove T-cell epitopes that derive from        hypervariable region sequences.    -   To remove most or all of the remaining T-cell epitopes,        mutations are introduced that, by the computer-based criteria        defined above, prevent binding of a peptide into a groove of an        MHC Class II molecule. In the case of antibody V regions, it is        preferable to introduce mutations that lie outside the CDRs        themselves, to avoid interfering with antigen binding.    -   In the case of fusion proteins of any type, it is generally the        case that the fusion junction will contain non-self T-cell        epitopes. These T-cell epitopes may be also removed by mutation.

As a specific embodiment the invention includes chimeric immunoglobulinsor fragments thereof wherein the reduced immunogenicity, the reducednumber of T-cell epitopes or the reduced number of peptides binding toMHC class II molecules is located to the target polypeptide portion X aswell as to the immunoglobulin portion or fragments thereof of thealtered fusion protein.

The invention includes also chimeric immunoglobulins as definedaccording to the invention wherein the immunoglobulin molecule and thenon-immunoglobulin target polypeptide (X) are fused via a linkermolecule (L). As a specific embodiment of the invention this linkermolecule itself has no or lower immunogenicity. Thus, the invention mayinclude immunoconjugates, wherein the linker molecule alone isde-immunized. Linker molecules which have reduced or no immunogenicityare known in the art or can be prepared by known methods or by themethod according to the invention. The invention also includes suchimmunoglobulin fusion proteins, wherein the immunoglobulin portion aswell as the target protein (X) portion of the fusion molecule andoptionally the linker molecule and the junction region (see below) areimmunogenicly modified. Alternatively, only one or more but not allportions of the molecule are modified according to the invention.

The invention relates, furthermore, to above-said immunoconjugates whichmay derive, in principal, from all immunoglobulin classes; however IgGis preferred. It is an object of the invention to provide such chimericimmunoglobulins which derive from IgG1, IgG2, IgG3 and IgG4. IgG1 andIgG2 immunoglobulins are preferred; IgG2 immunoglobulins are mostpreferred.

Since it has been shown that even recombinant proteins of human originand humanized antibodies may elicit an undesirable immune response inhumans it is object of this invention to provide fusion proteins whereinthe immunoglobulin portion as well as the target polypeptide portion (X)may be selected from non-human as well as from human origin. Sincehumanized or human-derived molecules have, as a rule, a less number ofT-cell epitopes, such molecules are preferred for de-immunization,because a less number of amino acid residues has to be modified.

Immunoconjugates (immunoglobulin (Ig) fusion proteins) according to theinvention include also fragments of antibodies like sFv, Fab, Fab′,F(ab′)2 and Fc. It is a specific and preferred object of the inventionto provide said above- and below-defined fusion proteins, wherein theimmunoglobulin portion is a Fc domain of an antibody, preferably an IgG1or IgG2 antibody. Fc-X molecules according to the invention which havereduced affinity to Fc receptors are a preferred object of theinvention. Fc molecules having a reduced affinity to Fc receptors arewell known in the art and can be prepared by modifying the amino acidsequence of the Fc domain (e.g. WO 99/43713).

In detail, the invention refers to:

-   -   an immunogenicly modified fusion protein derived from a parent        fusion protein, essentially consisting of a first        protein/polypeptide and a second protein/polypeptide, wherein        the first protein is an immunoglobulin molecule or a fragment        thereof and the second protein/polypetide is non-immunoglobulin        target polypeptide (X) each linked to the other directly or by a        linker molecule; said modified fusion protein having an amino        acid sequence different from that of said parent fusion protein        and exhibiting reduced immunogenicity by a reduced number of        T-cell epitopes within its amino acid sequence relative to the        parent fusion protein when exposed to the immune system of a        given species;    -   a corresponding fusion protein, wherein said T-cell epitopes are        peptide sequences able to bind to MCH class II molecule binding        groups;    -   a corresponding fusion protein, wherein the target        polypeptide (X) is linked by its N-terminal to the C-terminal of        the immunoglobulin moiety;    -   a correspondingly modified fusion protein, wherein the given        species is a human;    -   a corresponding fusion protein, wherein the fusion components        are fused via a linker molecule L;    -   a modified fusion protein according to claim 4, wherein said        linker molecule L is non-immunogenic or less immunogenic;    -   a corresponding fusion protein, wherein the junction region        represented by the C-terminal region of the immunoglobulin        portion and the N-terminal region of the non-immunoglobulin        target polypeptide (X) has no or a reduced number of T-cell        epitopes;    -   a corresponding fusion protein, wherein the immunoglobulin        portion or a fragment thereof or the target polypeptide (X)        portion is less immunogenic;    -   a corresponding fusion protein, wherein said immunoglobulin        molecule or fragment thereof is IgG1 or IgG2;    -   a corresponding fusion protein, wherein said immunoglobulin        fragment is a Fc portion, wherein, preferably, said Fc portion        has a reduced affinity to Fc receptors;    -   an immunogenicly modified fusion protein according having the        formula        Fc-L_(n)-X        -   wherein        -   Fc is the Fc portion of an immunoglobulin molecule            (antibody),        -   X is a non-immunoglobulin target polypeptide        -   L is a linker peptide,        -   n=0 or 1, and        -   wherein X and/or L comprises amino acid residue            modifications which elicit a reduced immunogenicity compared            to the parent molecule.        -   Preferred embodiments of these immunogenicly modified Fc            fusion molecules are:        -   Fc-X^(m), wherein X is modified only,        -   Fc-L^(m)-X^(m), wherein X and L are modified to have a            reduced immunogenicity,        -   Fc-X^(m) wherein X and the junction region between Fc and X            are modified,        -   Fc-L^(m)-X^(m), wherein X and L and the junction regions            between Fc, X and L are modified;    -   a corresponding Fc-(L)-X fusion protein wherein at least X is        immunogenicly modified;    -   an immunogenicly modified fusion protein having the formula        A-L_(n)-X        -   wherein        -   A is a whole antibody or its sFv, Fab, Fab′, F(ab′)₂            fragments        -   X is a non-immunoglobulin target polypeptide        -   L is a linker peptide,        -   n=0 or 1, and        -   wherein A and/or X and/or L comprise amino acid residue            modifications which elicit a reduced immunogenicity compared            to the parent molecule;        -   Preferred embodiments of these immunogenicly modified fusion            molecules are:        -   A-X^(m), wherein X is modified only, optionally the A-X            junction region,        -   A^(m)-X^(m), wherein A and X are modified, optionally their            junction region,        -   A-L^(m)-X^(m), wherein X and L are modified only to have a            reduced immunogenicity,        -   A^(m)-X, wherein A has reduced immunogenicity only,            optionally the A-X junction region,        -   A^(m)-L^(m)-X^(m), wherein A, L and X are immunogenicly            modified, optionally the A-L-X junction regions;    -   a corresponding A-(L)-X fusion molecule, wherein at least X or A        is immunogenicly modified;    -   a corresponding fusion protein, wherein A is selected from the        group:        -   anti-EGF receptor (HER1) antibodies        -   anti-HER2 antibodies        -   anti-CDx antibodies, wherein x is an integer from 1–25        -   anti-cytokine receptor antibodies        -   anti-17-1A antibodies,        -   anti-KSA antibodies        -   anti-GP IIb/IIa antibodies    -   anti-integrin receptor antibodies    -   anti VEGF receptor antibodies;    -   a correspondingly fusion protein, wherein the antibody is        selected from the group:        -   monoclonal antibody 225 and derivatives,        -   monoclonal antibody 425 and derivatives        -   monoclonal antibody KS 1/4 and derivatives        -   monoclonal antibody 14.18 and derivatives        -   monoclonal antibody 4D5/HER2 (Herceptin®) and derivatives        -   monoclonal antibody 17-1A and derivatives        -   monoclonal anti-CD3 antibodies        -   monoclonal antibody 7E3 and derivatives        -   monoclonal antibodies LM609, P1F6 and 14D9.F8 and            derivatives        -   monoclonal antibody DC-101 and derivatives        -   monoclonal anti-I1-2R antibody (Zenapax®) and derivatives    -   a corresponding fusion protein, wherein the target polypeptide X        is selected from the group:        -   cytokines, integrin inhibitors, soluble cytokine receptors,            glycoproteins, hormones, glycoprotein hormones, leptin,            growth hormones, growth factors, antihemophilic factors,            antigens, cytokine receptor antagonists;    -   a corresponding fusion protein, wherein the target polypeptide X        is selected from the group:        -   IL-2, G-CSF, GM-CSF, EPO, TPO, members of the interferon            family, TNFα, soluble TNF receptor, IL-12, IL-8, factor            VIII, FGF, TGF, EGF, VEGF, PMSA, IGF, insulin, RGD-peptides,            endostatin, angiostatin, BDNF, CNTF, protein c, factor VIII            and IX, and and biologically active fragments thereof;    -   a more specified corresponding fusion protein selected from the        group:        -   MAb KS 1/4-IL2, MAb 14.18-IL2        -   MAb 425-IL2, MAb c425-IL2, MAb h425-IL2, MAb 425-TNFa        -   MAb 225-IL2, MAb c225-IL2        -   MAb 4D5-IL2, MAb DC 101-I12, MAb LM609-IL2,        -   Fc-IL2, Fc-TNFa, Fc-G-CSF, Fc-EPO, Fc-Leptin, Fc-KGF,        -   Fc-BFNF, FC-β-Cerebrosidase, Fc-TPO, Fc-GM-CSF;    -   an immunogenicly modified artificial protein selected from the        group:        -   (i) Y-(L)-X, wherein Y is a cytokine and X, (L) is a            molecule as defined above,        -   (ii) P-(L)-X, wherein P is a protein with unusual            glycosylation moieties and X, (L) is a molecule as defined            above,        -   (iii) A-(L)-X, wherein A, X (L) is a molecule as defined            above, derived from a parent artificial protein having an            amino acid sequence which is different from that of said            parent artificial protein and exhibits reduced            immunogenicity by a reduced number of T-cell epitopes            relative to the parent fusion protein when exposed to the            immune system of a given species, wherein said T-cell            epitopes are peptide sequences able to bind to MCH class II            molecule binding groups obtainable or obtained by a method            as specified in this invention;    -   a DNA sequence encoding any fusion protein as specified above        and below;    -   a DNA sequence encoding a corresponding fusion protein,        comprising        -   (i) a signal sequence        -   (ii) a DNA sequence encoding all domains or a Fc, sFV, Fab,            Fab′ or F(ab′)2 domain of an IgG1, IgG2 or IgG3 antibody,            and        -   (ii) a DNA sequence encoding the polypeptide (X), and            optionally        -   (iii) a DNA sequence encoding the linker molecule;    -   an expression vector comprising a corresponding DNA sequence;    -   a pharmaceutical composition comprising a fusion protein as        specified above and below, optionally together with a suitable        carrier, excipient or diluent or another therapeutically        effective drug, such as chemotherapeutics or cytotoxic drugs;    -   a method for preparing an immunogenicly modified fusion protein        as specified comprising the steps:        -   (i) determining the amino acid sequence of the parent fusion            protein or part thereof;        -   (ii) identifying one or more potential T-cell epitopes            within the amino acid sequence of the fusion protein by any            method including determination of the binding of the            peptides to MHC molecules using in vitro or in silico            techniques or biological assays, (iii) designing new            sequence variants by alteration of at least one amino acid            residue within the originally identified T-cell epitope            sequences, said variants are modified in such a way to            substantially reduce or eliminate the activity or number of            the T-cell epitope sequences and/or the number of MHC            allotypes able to bind peptides derived from said biological            molecule as determined by the binding of the peptides to MHC            molecules using in vitro or in silico techniques or            biological assays or by binding of peptide-MHC complexes to            T-cells, (iv) constructing such sequence variants by            recombinant DNA techniques and testing said variants in            order to identify one or more variants with desirable            properties, and (v) optionally repeating steps (ii)–(iv),            characterized in that the identification of T-cell epitope            sequences according to step (ii) is achieved by        -   (a) selecting a region of the peptide having a known amino            acid residue sequence;        -   (b) sequentially sampling overlapping amino acid residue            segments of predetermined uniform size and constituted by at            least three amino acid residues from the selected            region; (c) calculating MHC Class II molecule binding score            for each said sampled segment by summing assigned values for            each hydrophobic amino acid residue side chain present in            said sampled amino acid residue segment; and (d) identifying            at least one of said segments suitable for modification,            based on the calculated MHC Class II molecule binding score            for that segment, to change overall MHC Class II binding            score for the peptide without substantially the reducing            therapeutic utility of the peptide;    -   a corresponding method, wherein step (c) is carried out by using        a Böhm scoring function modified to include 12-6 van der Waal's        ligand-protein energy repulsive term and ligand conformational        energy term by (1) providing a first data base of MHC Class II        molecule models; (2) providing a second data base of allowed        peptide backbones for said MHC Class II molecule models; (3)        selecting a model from said first data base; (4) selecting an        allowed peptide backbone from said second data base; (5)        identifying amino acid residue side chains present in each        sampled segment; (6) determining the binding affinity value for        all side chains present in each sampled segment; and        optionally (7) repeating steps (1) through (5) for each said        model and each said backbone;    -   a corresponding method, wherein the sampled amino acid residue        segment is constituted by 13 amino acid residues and/or        consecutive sampled amino acid residue segments overlap by one        to five amino acid residues;    -   a corresponding method, wherein 1–9 amino acid residues,        preferably one amino acid residue, in any of the originally        present T-cell epitope sequences (is) are altered;    -   a corresponding method, wherein the alteration of the amino acid        residues is substitution, deletion or addition of originally        present amino acid(s) residue(s) by other amino acid residue(s)        at specific position(s);    -   a corresponding method, wherein additionally further alteration        by substitution, deletion or addition is conducted to restore        biological activity of said biological molecule.

The polypeptides according to the invention include also antigens, likePMSA and others. Antigens which elicit a not desired and too strongimmune response can be modified according to the method of the inventionand result in antigens which have a reduced immunogenicity which ishowever strong enough for using the antigen e.g. as vaccine. Theinvention includes also variants and other modification of a specificpolypeptide, protein, fusion protein, immunoglobulin or immunoconjugatewhich have in principal the same biological activity and a similar(reduced) immunogenicity. All proteins mentioned above are well knownand described in the art or are already commercially available. Most ofthem are known to have a proved therapeutic benefit. The leader orsignal sequences and linker sequences may be optional.

Preparing the fusion protein by linking the immunoglobulin component byits C-terminus or its fragment to the N-terminus of thenon-immunoglobulin target polypeptide (X), optionally via the linkermolecule according to step (ii) as described above, is carried out by:

-   (i) preparing a gene construct comprising a DNA sequence encoding    the polypeptide X, a DNA sequence encoding the immunoglobulin    molecule or fragments thereof [sFv, Fab, Fab′, F(ab′)₂, Fc], and    optionally the DNA sequence of a the linker molecule, and-   (ii) expressing the gene construct by an expression system.

The immunoconjugates according to the present invention reveal enhancedproperties. Thus decreased protein degradation, increased stability andenhanced serum circulation half-life can be measured as well as adistinctly reduced immunogenicity and/or allergenicity.

Surprisingly, the reduced immunogenicity leads in many cases to afurther increase of half-life, especially in cases where Fc-X moleculesaccording to the invention are used. The reduced immunogenicity makesthe fusion proteins according to the invention more tolerable for agiven species compared to the non-modified fusion proteins and,therefore, can be administered in higher dosages if necessary.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates one of the mechanisms by which fusion proteinsdisplays enhanced immunogenicity. FIG. 1 a shows a protein (“X”) fusedto an Fc moiety binding to a cell bearing an Fc receptor. FIG. 1 b showsthe fusion protein being processed such that the “X” moiety ispreferentially degraded. FIG. 1 c shows a peptide remnant of “X” beingpresented by an MHC molecule to a T-cell.

FIG. 2 shows a mechanism of enhanced immunogenicity for a fusion proteincomprising an Fc moiety and a second moiety. FIG. 2 a shows the bindingof the fusion protein to a B cell that expresses an antibody specificfor “X” on its surface. The fusion protein is bound by both the specificantibody and by Fc receptors that are not already bound by the antibody.

FIG. 3 illustrates a second mechanism by which fusion proteins displaysenhanced immunogenicity. FIG. 1 a shows a protein (“X”) fused to ancytokine moiety binding to a B cell with a surface-bound antibody. FIG.3 b shows the fusion protein being processed. FIG. 3 c shows a peptideremnant of “X” being presented by an MHC molecule to a T-cell, at thesame time that additional X-cytokine fusion protein is bound to thesurface of the B cell.

FIG. 4 illustrates another mechanism by which an engineered proteindisplays enhanced immunogenicity. In this case, a cytokine-X fusionprotein directly activates a B cell. The B cell synthesizes a specificantibody to X, which increases the local concentration of the cytokinein the neighborhood of the B cell.

FIG. 5 illustrates another mechanism by which an engineered proteindisplays enhanced immunogenicity. FIG. 5 a shows the binding of aprotein bearing a glycosylation moiety to a specific cell-surfacereceptor for that glycosylation moiety on an immune cell. FIG. 5 b showsthe uptake and degradation of the glycosylated protein. FIG. 5 c showsthe presentation of a peptide remnant of the glycosylated protein to aT-cell via an MHC molecule.

FIG. 6 shows a mechanism by which an antibody-cytokine fusion proteindisplays enhanced immunogenicity. FIG. 6 a shows the binding of theantibody-cytokine fusion protein to a B cell that expresses an antibodyspecific for the CDRs of the antibody-cytokine fusion protein. Thefusion protein is bound by both the specific antibody and by Fcreceptors that are not already bound by the antibody. FIG. 6 b shows thefusion protein being processed. FIG. 6 c shows a peptide remnant of theCDRs being presented by an MHC molecule to a T-cell, at the same timethat additional antibody-cytokine fusion protein is bound to the surfaceof the B cell.

DETAILED DESCRIPTION OF THE INVENTION

The term “T-cell epitope” means according to the understanding of thisinvention an amino acid sequence which is able to bind with reasonableefficiency MHC class II molecules (or their equivalent in a non-humanspecies), able to stimulate T-cells and/or also to bind (withoutnecessarily measurably activating) T-cells in complex with MHC class II.

The term “peptide” as used herein and in the appended claims, is acompound that includes two or more amino acids. The amino acids arelinked together by a peptide bond (defined herein below). There are 20different naturally occurring amino acids involved in the biologicalproduction of peptides, and any number of them may be linked in anyorder to form to a peptide chain or ring. The naturally occurring aminoacids employed in the biological production of peptides all have theL-configuration. Synthetic peptides can be prepared employingconventional synthetic methods, utilizing L-amino acids, D-amino acids,or various combinations of amino acids of the two differentconfigurations. Some peptides contain only a few amino acid units. Shortpeptides, e.g., having less than ten amino acid units, are sometimesreferred to as “oligopeptides”. Other peptides contain a large number ofamino acid residues, e.g. up to 100 or more, and are referred to as“polypeptides”. By convention, a “polypeptide” may be considered as anypeptide chain containing three or more amino acids, whereas a“oligopeptide” is usually considered as a particular type of “short”polypeptide. Thus, as used herein, it is understood that any referenceto a “polypeptide” also includes an oligopeptide. Further, any referenceto a “peptide” includes polypeptides, oligopeptides, and proteins. Eachdifferent arrangement of amino acids forms different polypeptides orproteins. The number of polypeptides—and hence the number of differentproteins—that can be formed is practically unlimited.

The term “less or reduced immunogenic(ity)” used before and thereafteris a relative term and relates to the immunogenicity of the respectiveoriginal source molecule when exposed in vivo to the same type ofspecies compared with the molecule modified according to the invention.

The term “modified protein” as used according to this inventiondescribes a protein which has reduced number of T-cell epitopes andelicits therefore a reduced immunogenicity relative to the parentprotein when exposed to the immune system of a given species.

The term “non-modified protein” as used according to this inventiondescribes the “parent” protein as compared to the “modified protein” andhas a larger number of T-cell epitopes and, therefore, an enhancedimmunogenicity relative to the modified protein when exposed to theimmune system of a given species.

The term “biologically active protein” as used here and in the claimsincludes according to the invention polypetides, proteins,immunoglobulins such as antibodies, antibody fragments, fusion proteins,enzymes, antigens and so on, if not defined otherwise, which elicit abiological and/or therapeutic effect.

The term “cytokine” is used herein to describe proteins, analogsthereof, and fragments thereof which are produced and excreted by acell, and which elicit a specific response in a cell which has areceptor for that cytokine. Preferably, cytokines include interleukinssuch as interleukin-2 (IL-2), hematopoietic factors such asgranulocyte-macrophage colony stimulating factor (GM-CSF), tumornecrosis factor (TNF) such as TNFa, and lymphokines such as lymphotoxin.Preferably, the antibody-cytokine fusion protein of the presentinvention displays cytokine biological activity. In principal, theinventions encompasses all cytokines as recently classified according totheir receptor code (Inglot, 1997, Archivum Immunologiae et TherapiaeExperimentalis, 45: 353).

The phrase “single chain Fv” or “scFv” refers to an antibody in whichthe heavy chain and the light chain of a traditional two chain antibodyhave been joined to form one chain. Typically, a linker peptide isinserted between the two chains to allow for proper folding and creationof an active binding site.

The term “Fc region” or “Fc domain” as used in this invention isunderstood to mean the carboxyl terminal portion of an immunoglobulinheavy chain constant region, or an analog or portion thereof capable ofbinding an Fc receptor. As is known, each immunoglobulin heavy chainconstant region comprises four or five domains. The domains are namedsequentially as follows: CH1-hinge-CH2-CH3(-CH4). CH4 is present in IgM,which has no hinge region. The immunoglobulin heavy chain constantregion useful in the practice of the invention preferably comprises animmunoglobulin hinge region, and preferably also includes a CH3 domain.The immunoglobulin heavy chain constant region most preferably comprisesan immunoglobulin hinge region, a CH2 domain and a CH3 domain. Thepreferred Fc domain according to this invention consists thus of thehinge-CH2-CH3 domain.

As used herein, the term immunoglobulin “hinge region” is understood tomean an entire immunoglobulin hinge region or at least a portion of theimmunoglobulin hinge region sufficient to form one or more disulfidebonds with a second immunoglobulin hinge region.

As used herein, the term “signal sequence” is understood to mean asegment which directs the secretion of the fusion protein and thereafteris cleaved following translation in the host cell. The signal sequenceof the invention is a polynucleotide which encodes an amino acidsequence which initiates transport of a protein across the membrane ofthe endoplasmic reticulum. Signal sequences which are useful in theinvention include antibody light chain signal sequences, e.g., antibody14.18 (Gillies et. al. (1989) J. Immunol. Meth. 125: 191), and any othersignal sequences which are known in the art (see, e.g., Watson, 1984,Nucleic Acid Research 12:5145).

The term “mutant or variant” used with respect to a particular proteinencompasses any molecule such as a truncated or other derivative of therelevant protein which retains substantially the same activity in humansas the relevant protein. Such other derivatives can be prepared by theaddition, deletion, substitution, or rearrangement of amino acids or bychemical modifications thereof.

It is contemplated that suitable immunoglobulin heavy chain constantregions may be derived from antibodies belonging to each of theimmunoglobulin classes referred to as IgA, IgD, IgE, IgG, and IgM,however, immunoglobulin heavy chain constant regions from the IgG classare preferred.

Furthermore, it is contemplated that immunoglobulin heavy chain constantregions may be derived from any of the IgG antibody subclasses referredto in the art as IgG1, IgG2, IgG3, and IgG4. Immunoglobulin heavy chainconstant region domains have cross-homology among the immunoglobulinclasses. For example, the CH2 domain of IgG is homologous to the CH2domain of IgA and IgD, and to the CH3 domain of IgM and IgE. The choiceof appropriate immunoglobulin heavy chain constant regions is discussedin detail in U.S. Pat. No. 5,541,087 and U.S. Pat. No. 5,726,044. Thechoice of particular immunoglobulin heavy chain constant regionsequences from certain immunoglobulin classes and subclasses to achievea particular result is considered to be within the level of skill in theart. It may be useful, in some circumstances, to modify theimmunoglobulin heavy chain constant region, for example, by mutation,deletion or other changes mediated by genetic engineering or otherapproaches, so that certain activities, such as complement fixation orstimulation of antibody-dependent cell-mediated cytotoxicity (ADCC) arereduced or eliminated.

The Fc region is considered non- or weakly immunogenic if theimmunoglobulin heavy chain constant region fails to generate adetectable antibody response.

Furthermore, it is contemplated that substitution or deletion of aminoacids within the immunoglobulin heavy chain constant regions may beuseful in the practice of the invention. One example may includeintroducing amino acid substitutions in the upper CH2 region to create aFc variant with reduced affinity for Fc receptors (Cole et al. (1997) J.Immunol. 159:3613). An antibody-based fusion protein with an enhanced invivo circulating half-life can be obtained by constructing a fusionprotein having reduced binding affinity for a Fc receptor, and avoidingthe use of sequences from antibody isotypes that bind to Fc receptors(WO 99/43713). For example, of the four known IgG isotypes, IgG1 (Cγ1)and IgG3 (Cγ3) are known to bind FcRγ1 with high affinity, whereas IgG4has a 10-fold lower binding affinity, and IgG2 (Cγ2) does not bind toFcRγ1. Thus, an antibody-based fusion protein with reduced bindingaffinity for a Fc receptor could be obtained by constructing a fusionprotein with a Cγ2 constant region (Fc region) or a Cγ4 Fc region, andavoiding constructs with a Cγ1 Fc region or a Cγ3 Fc region. Anantibody-based fusion protein with an enhanced in vivo circulatinghalf-life can be obtained by modifying sequences necessary for bindingto Fc receptors in isotypes that have binding affinity for an Fcreceptor, in order to reduce or eliminate binding.

The important sequences for FcγR binding are Leu-Leu-Gly-Gly (residues234 through 237 in Cγ1), located in the CH2 domain adjacent to the hinge(Canfield and Morrison, J. Exp. Med. 173: 1483–1491 (1991)). Anotherimportant structural component necessary for effective FcR binding isthe presence of an N-linked carbohydrate chain covalently bound toAsn₂₉₇. Enzymatic removal of this structure or mutation of the Asnresidue effectively abolish, or at least dramatically reduce, binding toall classes of FcγR.

The resulting antibody-based fusion proteins have a longer in vivocirculating half-life than the unlinked second non-immunoglobulinprotein. Dimerization of a ligand can increase the apparent bindingaffinity between the ligand and its receptor. For instance, if one Xmoiety of an Fc-X fusion protein can bind to a receptor on a cell with acertain affinity, the second X moiety of the same Fc-Interferon-alphafusion protein may bind to a second receptor on the same cell with amuch higher avidity (apparent affinity). This may occur because of thephysical proximity of the second X moiety to the receptor after thefirst X moiety already is bound. In the case of an antibody binding toan antigen, the apparent affinity may be increased by at least tenthousand-fold. Each protein subunit, i.e., “X,” has its own independentfunction so that in a multivalent molecule, the functions of the proteinsubunits may be additive or synergistic. Thus, fusion of the normallydimeric Fc molecule or another antibody fragment to a polypeptide X mayincrease the activity of X.

Nucleic acid sequences encoding, and amino acid sequences defining ahuman immunoglobulin Fc region, especially a Fcγ1, Fcγ2 and Fcγ3, usefulin the practice of the invention are set forth in in the prior, such asdisclosed in (WO 00/40615, WO 00/69913, WO 00/24782) or in the Genbankand/or EMBL databases, for example, AF045536.1 (Macaca fuscicularis),AF045537.1 (Macaca mulatta), AB016710 (Felix catus), K00752 (Oryctolaguscuniculus), U03780 (Sus scrofa), 248947 (Camelus dromedarius), X62916(Bos taurus), L07789 (Mustela vison), X69797 (Ovis aries), U17 166(Cricetulus migratorius), X07189 (Rattus rattus), AF57619.1 (Trichosurusvulpecula), or AF035195 (Monodelphis domestica).

Thus, vectors reported earlier (Lo et al. (1998) Protein Engineering11:495–500) were modified by replacing the human IgG1 Fc sequence withsequences from cDNA encoding the mouse IgG2a Fc (U.S. Pat. No.5,726,044).

The invention encompasses mutations in the immunoglobulin componentwhich eliminate undesirable properties of the native immunoglobulin,such as Fc receptor binding and/or introduce desirable properties suchas stability. For example, Angal S., King D. J., Bodmer M. W., TurnerA., Lawson A. D. G., Roberts G., Pedley B. and Adair R., MolecularImmunology, 130, pp 105–108, 1993, describe an IgG4 molecule whereresidue 241 (Kabat numbering) is altered from serine to proline. Thischange increases the serum half-life of the IgG4 molecule. Canfield S.M. and Morrison S. L., Journal of Experimental Medicine vol 173 pp1483–1491, describe the alteration of residue 248 (Kabat numbering) fromleucine to glutamate in IgG3 and from glutamate to leucine in mouseIgG2b. Substitution of leucine for glutamate in the former decreases theaffinity of the immunoglobulin molecule concerned for the FcγR1receptor, and substitution of glutamate for leucine in the latterincreases the affinity. EP 0307 434 discloses various mutationsincluding an L to E mutation at residue 248 (Kabat numbering) in IgG.The constant domain(s) or fragment thereof is preferably the whole or asubstantial part of the constant region of the heavy chain of human IgG.The IgG component suitably comprises the CH2 and CH3 domains and thehinge region including cysteine residues contributing to inter-heavychain disulphide bonding. For example when the IgG component is derivedfrom IgG4 it includes cysteine residues 8 and 11 of the IgG4 hingeregion (Pinck J. R. and Milstein C., Nature, 121, 6 pp 941–942, 1967).

The process of the invention may be performed by conventionalrecombinant techniques such as described in Maniatis et. al. (MolecularCloning—A Laboratory Manual; Cold Spring Harbor, 1982) and DNA CloningVols I, II and III (D. M. Glover ed., IRL Press Ltd) or Sambrook et al.(1989, Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, NY, USA).

In particular, the process may comprise the steps of:

-   (i) preparing a replicable expression vector capable, in a host    cell, of expressing a DNA polymer comprising a nucleotide sequence    that encodes said compound;-   (ii) transforming a host cell with said vector;-   (iii) culturing said transformed host cell under conditions    permitting expression of said DNA polymer to produce said compound;    and-   (iv) recovering said compound.

The invention also provides a process for preparing the DNA polymer bythe condensation of appropriate mono-, di- or oligomeric nucleotideunits. The preparation may be carried out chemically, enzymatically, orby a combination of the two methods, in virro or in vivo as appropriate.Thus, the DNA polymer may be prepared by the enzymatic ligation ofappropriate DNA fragments, by conventional methods such as thosedescribed by D. M. Roberts et al in Biochemistry 1985, 24, 5090–5098.The DNA fragments may be obtained by digestion of DNA containing therequired sequences of nucleotides with appropriate restriction enzymes,by chemical synthesis, by enzymatic polymerisation on DNA or RNAtemplates, or by a combination of these methods. Digestion withrestriction enzymes may be performed in an appropriate buffer at atemperature of 20°–70° C. with 0.1–10 μg DNA. Enzymatic polymerisationof DNA may be carried out in vitro using a DNA polymerase such as DNApolymerase I (Klenow fragment) in an appropriate buffer containing thenucleoside uiphosphates DATP, dCTP, dGTP and dITP as required at atemperature of 10°–37° C., generally in a volume of 50 μl or less.Enzymatic ligation of DNA fragments may be carried out using a DNAligase such as T4 DNA ligase in an appropriate buffer at a temperatureof 40° C. to ambient, generally in a volume of 50 μl or less.

The chemical synthesis of the DNA polymer or fragments may be carriedout by conventional phosphotriester, phosphite or phosphoramiditechemistry, using solid phase techniques such as those described in“Chemical and Enzymatic Synthesis of Gene Fragments—A Laboratory Manual”(ed. H. G. Gassen and A. Lang), Verlag Chemie, Weinheim (1982), or inother scientific publications, for example M. J. Gait, H. W. D. Matthes,M. Singh, B. S. Sproat, and R. C. Titmas, Nucleic Acids Research, 1982,10, 6243; B. S. Sproat and W. Bannwarth, Tetrahedron Letters, 1983, 24,5771; M. D. Matteucci and M. H Caruthers, Tetrahedron Letters, 1980, 21,719; M. D. Matteucci and M. H. Caruthers, Journal of the AmericanChemical Society, 1981, 103, 3185; S. P. Adams et al., Journal of theAmerican Chemical Society, 1983, 105, 661; N. D. Sinha, J. Biemat, J.McMannus, and H. Koester, Nucleic Acids Research, 1984, 12, 4539; and H.W. D. Matthes et al., EMBO Journal, 1984, 3, 801. Preferably anautomated DNA synthesizer is employed.

The DNA molecules may be obtained by the digestion with suitablerestriction enzymes of vectors carrying the required coding sequences orby use of polymerase chain reaction technology. The precise structure ofthe DNA molecules and the way in which they are obtained depends uponthe structure of the desired product. The design of a suitable strategyfor the construction of the DNA molecule coding for the compound is aroutine matter for the skilled worker in the art.

The expression of the DNA polymer encoding the compound in a recombinanthost cell may be carried out by means of a replicable expression vectorcapable, in the host cell, of expressing the DNA polymer. The expressionvector is novel and also forms part of the invention. The replicableexpression vector may be prepared in accordance with the invention, bycleaving a vector compatible with the host cell to provide a linear DNAsegment having an intact replicon, and combining said linear segmentwith one or more DNA molecules which, together with said linear segment,encode the compound, under ligating conditions. The ligation of thelinear segment and more than one DNA molecule may be carried outsimultaneously or sequentially as desired. Thus, the DNA polymer may bepreformed or formed during the construction of the vector, as desired. Auseful expression vector is described at Lo et al. (1988) ProteinEngineeering 11:495, in which the transcription of the Fc-X geneutilizes the enhancer/promoter of the human cytomegalovirus and the SV40polyadenylation signal. Suitable vectors include plasmids,bacteriophages, cosmids and recombinant viruses derived from, forexample, baculoviruses, vaccinia or Semliki Forest virus. Thus, vectorsreported earlier (Lo et al. (1998) Protein Engineering 11:495–500) weremodified by replacing the human IgG1 Fc sequence with sequences fromcDNA encoding the mouse IgG2a Fc (U.S. Pat. No. 5,726,044).

The choice of vector will be determined in part by the host cell, whichmay be prokaryotic, such as E. coli, or eukaryotic, such as mouse C127,mouse myeloma, Chinese hamster ovary, COS or Hela cells, fungi e.g.filamentous fungi or unicellular yeast or an insect cell such asDrosophila. Currently preferred host cells for use in the inventioninclude immortal hybridoma cells, NS/0 myeloma cells, 293 cells, Chinesehamster ovary cells, HELA cells, and COS cells. The host cell may alsobe a transgenic animal.

Polymerisation and ligation may be performed as described above for thepreparation of the DNA polymer. Digestion with restriction enzymes maybe performed in an appropriate buffer at a temperature of 20°–70° C.with 0.1–10 μg DNA. The recombinant host cell is prepared, in accordancewith the invention, by transforming a host cell with a replicableexpression vector of the invention under transforming conditions.Suitable transforming conditions are conventional and are described in,for example, Maniatis et al., cited above, or “DNA Cloning” Vol. II, D.M. Glover ed., IRL Press Ltd, 1985. The choice of transformingconditions is determined by the host cell. Thus, a bacterial host suchas E. coli may be treated with a solution of CaCl₂ (Cohen er al, Proc.Nat. Acad. Sci., 1973, 69, 2110) or with a solution comprising a mixtureof RbCl, MnCl₂, potassium acetate and glycerol, and then with3-[N-morpholinol-propane-sulphonic acid, RbCl and glycerol. Mammaliancells in culture may be transformed by calcium co-precipitation of thevector DNA onto the cells. The invention also extends to a host celltransformed or transfected with a replicable expression vector of theinvention. Culturing the transformed host cell under conditionspermitting expression of the DNA polymer is carried out conventionally,as described in, for example, Maniatis et al. and “DNA Cloning” citedabove. Thus, preferably the cell is supplied with nutrient and culturedat a temperature below 45° C. The expression product is recovered byconventional methods according to the host cell. Thus, where the hostcell is bacterial, such as E. coli it may be lysed physically,chemically or enzymatically and the protein product isolated from theresulting lysate. If the product is to be secreted from the bacterialcell it may be recovered from the periplasmic space or the nutrientmedium. Where the host cell is mammalian, the product may generally beisolated from the nutrient medium. The DNA polymer may be assembled intovectors designed for isolation of stable transfomed mammalian cell linesexpressing the product: e.g. bovine papillomavirus vectors or amplifiedvectors in Chinese hamster ovary cells (DNA cloning Vol.11 D. M. Glovered. IRL Press 1985; Kaufman, R. J., Molecular and Cellular Biology 5,1750–1759, 1985; Pavlakis G. N. and Hamer, D. H., Proceedings of theNational Academy of Sciences (USA) 80, 397–401, 1983; Goeddel, D. V. etal., and EP 0 093 619, 1983).

The immunoconjugates of the invention may comprise linker molecules. Thelinker is preferably made up of amino acids linked together by peptidebonds. Thus, in preferred embodiments, the linker is made up of from 1to 20 amino acids linked by peptide bonds, wherein the amino acids areselected from the 20 naturally occurring amino acids. Some of theseamino acids may be glycosylated, as is well understood by those in theart. In a more preferred embodiment, the 1 to 20 amino acids areselected from glycine, alanine, proline, asparagine, glutamine, andlysine. Even more preferably, a linker is made up of a majority of aminoacids that are sterically unhindered, such as glycine and alanine. Thus,preferred linkers are polyglycines such as

-   polyGly (particularly (Gly)₂-(Gly)₇),-   poly(Gly-Ala),-   polyAla.

Other specific examples of suitable linkers are:

-   (Gly)₃Lys(Gly)₄ (SEQ ID NO: 1),-   (Gly)₃AsnGlySer(Gly)₂ (SEQ ID NO: 2),-   (Gly)₃Cys(Gly)₄ (SEQ ID NO: 3), and-   GlyProAsnGlyGly (SEQ ID NO: 4).

Combinations of Gly and Ala are also preferred. The linkers shown hereare exemplary; linkers within the scope of this invention may be muchlonger and may include other residues. Non-peptide linkers are alsopossible. The peptide linkers may be altered to form derivatives in thesame manner as described above.

Preferred linkers of the invention are not or less immunogenic. Most ofthe above-cited linker peptides are at least less immunogenic. Howeverit is possible that creating the linkage between an antibody or a sFv,Fab, Fab′ or F(ab′)2 or a Fc domain and the target protein via a linkerpeptide molecule as mentioned above, new immunogenic epitopes may benewly created within the linkage region resulting in an immunoconjugatewhich has an increased immunogenicity compared to the immunogenicity ofthe single (de-immunized) components. This situation can also beextended to fusion protein having no linker molecule. Therefore, theinvention also relates to de-immunized regions of a fusion proteinaccording to the invention, the so-called fusion or junction regions.When fusing a first protein molecule with a second protein molecule(which also can be a linker molecule) via the C- and N-terminals asequence region is created that is artificial and, thus, was usually notyet seen by the immune system. This region is deemed is be immunogenic.The region of amino acid residues comprise according to the inventionapproximately 10 residues of each protein terminal (N- or C-terminal).The complete fusion region comprises, therefore, about 20 amino acidresidues, preferably 2–16, more preferably 2–10 (which is 1–8 and 1–5amino acid residues, respectively, of each fusion partner).

The invention includes also further Fc variants. Such further Fcvariants, one may remove one or more sites of a native Fc that providestructural features or functional activity not required by the fusionmolecules of this invention. One may remove these sites by, for example,substituting or deleting residues, inserting residues into the site, ortruncating portions containing the site. The inserted or substitutedresidues may also be altered amino acids, such as peptidomimetics orD-amino acids. For example, one or more glycosylation sites may beremoved. Residues that are typically glycosylated (e.g., asparagine) mayconfer cytolytic response. Such residues may be deleted or substitutedwith unglycosylated residues (e.g., alanine). ADCC site as well as sitesinvolved in interaction with complement, such as the Clq binding site,may also be removed if there is a specific need.

The invention includes also derivatives of the target polypeptide (X) ofthe invention. Such derivatives may further improve the solubility,absorption, biological half life, and the like of (X). The modified (X)may alternatively eliminate or attenuate any undesirable side-effect andthe like. Exemplary derivatives include also compounds in which (X) orsome portion thereof is cyclic. For example, the peptide portion may bemodified to contain two or more Cys residues (e.g., in the linker),which could cyclize by disulfide bond formation. The compound iscross-linked or is rendered capable of cross-linking between molecules.For example, the peptide portion may be modified to contain one Cysresidue and thereby be able to form an intermolecular disulfide bondwith a like molecule.

In a final aspect the present invention relates to pharmaceuticalcompositions comprising said biologically active proteins obtainable bythe methods disclosed in the present invention, and methods fortherapeutic treatment of humans using the modified molecules andpharmaceutical compositions.

Therapeutic compositions of the present invention contain aphysiologically tolerable carrier together with the relevant agent asdescribed herein, dissolved or dispersed therein as an activeingredient. As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike. The preparation of a pharmacological composition that containsactive ingredients dissolved or dispersed therein is well understood inthe art and need not be limited based on formulation. Typically, suchcompositions are prepared as injectables either as liquid solutions orsuspensions, however, solid forms suitable for solution, or suspensions,in liquid prior to use can also be prepared. The preparation can also beemulsified. The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipients are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof. In addition, ifdesired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like which enhance the effectiveness of the active ingredient.

The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.Particularly preferred is the HCl salt when used in the preparation ofcyclic polypeptide αv antagonists. Physiologically tolerable carriersare well known in the art. Exemplary of liquid carriers are sterileaqueous solutions that contain no materials in addition to the activeingredients and water, or contain a buffer such as sodium phosphate atphysiological pH value, physiological saline or both, such asphosphate-buffered saline. Still further, aqueous carriers can containmore than one buffer salt, as well as salts such as sodium and potassiumchlorides, dextrose, polyethylene glycol and other solutes. Liquidcompositions can also contain liquid phases in addition to and to theexclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions.

Typically, a therapeutically effective amount of a modifiedimmunoglobulin in the form of an modified antibody or antibody fragmentaccording to the invention is an amount such that when administered inphysiologically tolerable composition is sufficient to achieve a plasmaconcentration of from about 0.01 microgram (μg) per milliliter (ml) toabout 100 μg/ml, preferably from about 1 μg/ml to about 5 μg/ml andusually about 5 μg/ml. Stated differently, the dosage can vary fromabout 0.1 mg/kg to about 300 mg/kg, preferably from about 0.2 mg/kg toabout 200 mg/kg, most preferably from about 0.5 mg/kg to about 20 mg/kg,in one or more dose administrations daily for one or several days. Wherethe immunotherapeutic agent is in the form of a fragment of a monoclonalantibody or a conjugate, the amount can readily be adjusted based on themass of the fragment/conjugate relative to the mass of the wholeantibody. A preferred plasma concentration in molarity is from about 2micromolar (μM) to about 5 millimolar (mM) and preferably, about 100 μMto 1 mM antibody antagonist.

A therapeutically effective amount of an agent according of thisinvention which is a non-immunotherapeutic peptide or a protein istypically an amount of such a molecule such that when administered in aphysiologically tolerable composition is sufficient to achieve a plasmaconcentration of from about 0.1 microgram (μg) per milliliter (ml) toabout 200 μg/ml, preferably from about 1 μg/ml to about 150 μg/ml. Basedon a protein having a mass of about 500 grams per mole, the preferredplasma concentration in molarity is from about 2 micromolar (μM) toabout 5 millimolar (mM) and preferably about 100 μM to 1 mM polypeptideantagonist.

The pharmaceutical compositions of the invention can comprise phraseencompasses treatment of a subject with agents that reduce or avoid sideeffects associated with the combination therapy of the present invention(“adjunctive therapy”), including, but not limited to, those agents, forexample, that reduce the toxic effect of anticancer drugs. Saidadjunctive agents prevent or reduce the incidence of nausea and vomitingassociated with chemotherapy, radiotherapy or operation, or reduce theincidence of infection associated with the administration ofmyelosuppressive anticancer drugs. Adjunctive agents are well known inthe art. The modified proteins according to the invention canadditionally administered with adjuvants like BCG and immune systemstimulators.

Furthermore, the compositions may include immunotherapeutic agents,chemotherapeutic agents and anti-neoplastic agents which may containcytotoxic effective radio labeled isotopes, or other cytotoxic agents,such as a cytotoxic peptides (e.g. cytokines) or cytotoxic drugs and thelike. The typical dosage of an active agent, which is a preferably achemical antagonist or a (chemical) chemotherapeutic agent according tothe invention (neither an immunotherapeutic agent nor anon-immunotherapeutic peptide/protein) is 10 mg to 1000 mg, preferablyabout 20 to 200 mg, and more preferably 50 to 100 mg per kilogram bodyweight per day.

The following examples describe the in invention in more detail.However, this listing does not limit the invention.

EXAMPLE 1

The following example describes in detail a preferred method foridentification of immunogenic sequence regions (T-cell epitopes) withinthe sequences of the fusion proteins as disclosed in this invention.However, it should be pointed out, that said molecules can be obtainedby other known methods.

The identification of T-cell epitopes of the molecules which weremodified in order to obtain the immunoconjugates according to thepresent invention can be achieved by different methods which aredescribed in the prior art (WO 92/10755 and WO 96/40792 (Novo Nordisk),EP 0519 596 (Merck & Co.), EP 0699 755 (Centro de ImmunologiaMoelcular), WO 98/52976 and WO 98/59244 (Biovation Ltd.) or relatedmethods.

Advantageous immunoconjugates, however, can be obtained if theidentification of said epitopes is realized by the following new methodwhich is described herewith in detail:

There are a number of factors that play important roles in determiningthe total structure of a protein, polypeptide or immunoglobulin. First,the peptide bond, i.e., that bond which joins the amino acids in thechain together, is a covalent bond. This bond is planar in structure,essentially a substituted amide. An “amide” is any of a group of organiccompounds containing the grouping —CONH—.

The planar peptide bond linking Cα of adjacent amino acids may berepresented as depicted below:

Because the O═C and the C—N atoms lie in a relatively rigid plane, freerotation does not occur about these axes. Hence, a plane schematicallydepicted by the interrupted line is sometimes referred to as an “amide”or “peptide plane” plane wherein lie the oxygen (O), carbon (C),nitrogen (N), and hydrogen (H) atoms of the peptide backbone. Atopposite corners of this amide plane are located the Cα atoms. Sincethere is substantially no rotation about the O═C and C—N atoms in thepeptide or amide plane, a polypeptide chain thus comprises a series ofplanar peptide linkages joining the Cα atoms.

A second factor that plays an important role in defining the totalstructure or conformation of a polypeptide or protein is the angle ofrotation of each amide plane about the common Cα linkage. The terms“angle of rotation” and “torsion angle” are hereinafter regarded asequivalent terms. Assuming that the O, C, N, and H atoms remain in theamide plane (which is usually a valid assumption, although there may besome slight deviations from planarity of these atoms for someconformations), these angles of rotation define the N and Rpolypeptide's backbone conformation, i.e., the structure as it existsbetween adjacent residues. These two angles are known as φ and ψ. A setof the angles φ₁, ψ₁, where the subscript i represents a particularresidue of a polypeptide chain, thus effectively defines the polypeptidesecondary structure. The conventions used in defining the φ, ψ angles,i.e., the reference points at which the amide planes form a zero degreeangle, and the definition of which angle is φ, and which angle is ψ, fora given polypeptide, are defined in the literature. See, e.g.,Ramachandran et al. Adv. Prol. Chem. 23:283–437 (1968), at pages 285–94,which pages are incorporated herein by reference.

The present method can be applied to any protein, and is based in partupon the discovery that in humans the primary Pocket 1 anchor positionof MHC Class II molecule binding grooves has a well designed specificityfor particular amino acid side chains. The specificity of this pocket isdetermined by the identity of the amino acid at position 86 of the betachain of the MHC Class II molecule. This site is located at the bottomof Pocket 1 and determines the size of the side chain that can beaccommodated by this pocket. Marshall, K. W., J. Immunol., 152:4946–4956(1994). If this residue is a glycine, then all hydrophobic aliphatic andaromatic amino acids (hydrophobic aliphatics being: valine, leucine,isoleucine, methionine and aromatics being: phenylalanine, tyrosine andtryptophan) can be accommodated in the pocket, a preference being forthe aromatic side chains. If this pocket residue is a valine, then theside chain of this amino acid protrudes into the pocket and restrictsthe size of peptide side chains that can be accommodated such that onlyhydrophobic aliphatic side chains can be accommodated. Therefore, in anamino acid residue sequence, wherever an amino acid with a hydrophobicaliphatic or aromatic side chain is found, there is the potential for aMHC Class II restricted T-cell epitope to be present. If the side-chainis hydrophobic aliphatic, however, it is approximately twice as likelyto be associated with a T-cell epitope than an aromatic side chain(assuming an approximately even distribution of Pocket 1 typesthroughout the global population).

A computational method embodying the present invention profiles thelikelihood of peptide regions to contain T-cell epitopes as follows:

(1) The primary sequence of a peptide segment of predetermined length isscanned, and all hydrophobic aliphatic and aromatic side chains presentare identified. (2) The hydrophobic aliphatic side chains are assigned avalue greater than that for the aromatic side chains; preferably abouttwice the value assigned to the aromatic side chains, e.g., a value of 2for a hydrophobic aliphatic side chain and a value of 1 for an aromaticside chain. (3) The values determined to be present are summed for eachoverlapping amino acid residue segment (window) of predetermined uniformlength within the peptide, and the total value for a particular segment(window) is assigned to a single amino acid residue at an intermediateposition of the segment (window), preferably to a residue at about themidpoint of the sampled segment (window). This procedure is repeated foreach sampled overlapping amino acid residue segment (window). Thus, eachamino acid residue of the peptide is assigned a value that relates tothe likelihood of a T-cell epitope being present in that particularsegment (window). (4) The values calculated and assigned as described inStep 3, above, can be plotted against the amino acid coordinates of theentire amino acid residue sequence being assessed. (5) All portions ofthe sequence which have a score of a predetermined value, e.g., a valueof 1, are deemed likely to contain a T-cell epitope and can be modified,if desired.

This particular aspect of the present invention provides a generalmethod by which the regions of peptides likely to contain T-cellepitopes can be described. Modifications to the peptide in these regionshave the potential to modify the MHC Class II binding characteristics.According to another aspect of the present invention, T-cell epitopescan be predicted with greater accuracy by the use of a moresophisticated computational method which takes into account theinteractions of peptides with models of MHC Class II alleles.

The computational prediction of T-cell epitopes present within a peptideaccording to this particular aspect contemplates the construction ofmodels of at least 42 MHC Class II alleles based upon the structures ofall known MHC Class II molecules and a method for the use of thesemodels in the computational identification of T-cell epitopes, theconstruction of libraries of peptide backbones for each model in orderto allow for the known variability in relative peptide backbone alphacarbon (Cα) positions, the construction of libraries of amino-acid sidechain conformations for each backbone dock with each model for each ofthe 20 amino-acid alternatives at positions critical for the interactionbetween peptide and MHC Class II molecule, and the use of theselibraries of backbones and side-chain conformations in conjunction witha scoring function to select the optimum backbone and side-chainconformation for a particular peptide docked with a particular MHC ClassII molecule and the derivation of a binding score from this interaction.

Models of MHC Class II molecules can be derived via homology modelingfrom a number of similar structures found in the Brookhaven Protein DataBank (“PDB”). These may be made by the use of semi-automatic homologymodeling software (Modeller, Sali A. & Blundell T L., 1993. J. Mol Biol234:779–815) which incorporates a simulated annealing function, inconjunction with the CHARMm force-field for energy minimisation(available from Molecular Simulations Inc., San Diego, Calif.).Alternative modeling methods can be utilized as well.

The present method differs significantly from other computationalmethods which use libraries of experimentally derived binding data ofeach amino-acid alternative at each position in the binding groove for asmall set of MHC Class II molecules (Marshall, K. W., et al., Biomed.Pept. Proteins Nucleic Acids, 1(3):157–162) (1995) or yet othercomputational methods which use similar experimental binding data inorder to define the binding characteristics of particular types ofbinding pockets within the groove, again using a relatively small subsetof MHC Class II molecules, and then ‘mixing and matching’ pocket typesfrom this pocket library to artificially create further ‘virtual’ MHCClass II molecules (Stumiolo T., et al., Nat. Biotech, 17(6): 555–561(1999). Both prior methods suffer the major disadvantage that, due tothe complexity of the assays and the need to synthesize large numbers ofpeptide variants, only a small number of MHC Class II molecules can beexperimentally scanned. Therefore the first prior method can only makepredictions for a small number of MHC Class II molecules. The secondprior method also makes the assumption that a pocket lined with similaramino-acids in one molecule will have the same binding characteristicswhen in the context of a different Class II allele and suffers furtherdisadvantages in that only those MHC Class II molecules can be‘virtually’ created which contain pockets contained within the pocketlibrary. Using the modeling approach described herein, the structure ofany number and type of MHC Class II molecules can be deduced, thereforealleles can be specifically selected to be representative of the globalpopulation. In addition, the number of MHC Class II molecules scannedcan be increased by making further models further than having togenerate additional data via complex experimentation.

The use of a backbone library allows for variation in the positions ofthe Cα atoms of the various peptides being scanned when docked withparticular MHC Class II molecules. This is again in contrast to thealternative prior computational methods described above which rely onthe use of simplified peptide backbones for scanning amino-acid bindingin particular pockets. These simplified backbones are not likely to berepresentative of backbone conformations found in ‘real’ peptidesleading to inaccuracies in prediction of peptide binding. The presentbackbone library is created by superposing the backbones of all peptidesbound to MHC Class II molecules found within the Protein Data Bank andnoting the root mean square (RMS) deviation between the Cα atoms of eachof the eleven amino-acids located within the binding groove. While thislibrary can be derived from a small number of suitable available mouseand human structures (currently 13), in order to allow for thepossibility of even greater variability, the RMS figure for each C″-αposition is increased by 50%. The average Cα position of each amino-acidis then determined and a sphere drawn around this point whose radiusequals the RMS deviation at that position plus 50%. This sphererepresents all allowed Cα positions.

Working from the Cα with the least RMS deviation (that of the amino-acidin Pocket 1 as mentioned above, equivalent to Position 2 of the 11residues in the binding groove), the sphere is three-dimensionallygridded, and each vertex within the grid is then used as a possiblelocation for a Cα of that amino-acid. The subsequent amide plane,corresponding to the peptide bond to the subsequent amino-acid isgrafted onto each of these Cαs and the φ and ψ angles are rotatedstep-wise at set intervals in order to position the subsequent Cα. Ifthe subsequent Cα falls within the ‘sphere of allowed positions’ forthis Cα than the orientation of the dipeptide is accepted, whereas if itfalls outside the sphere then the dipeptide is rejected. This process isthen repeated for each of the subsequent Cα positions, such that thepeptide grows from the Pocket 1 Cα ‘seed’, until all nine subsequent Cαshave been positioned from all possible permutations of the precedingCαs. The process is then repeated once more for the single Cα precedingpocket 1 to create a library of backbone Cα positions located within thebinding groove.

The number of backbones generated is dependent upon several factors: Thesize of the ‘spheres of allowed positions’; the fineness of the griddingof the ‘primary sphere’ at the Pocket 1 position; the fineness of thestep-wise rotation of the φ and ψ angles used to position, subsequentCαs. Using this process, a large library of backbones can be created.The larger the backbone library, the more likely it will be that theoptimum fit will be found for a particular peptide within the bindinggroove of an MHC Class II molecule. Inasmuch as all backbones will notbe suitable for docking with all the models of MHC Class II moleculesdue to clashes with amino-acids of the binding domains, for each allelea subset of the library is created comprising backbones which can beaccommodated by that allele. The use of the backbone library, inconjunction with the models of MHC Class II molecules creates anexhaustive database consisting of allowed side chain conformations foreach amino-acid in each position of the binding groove for each MHCClass II molecule docked with each allowed backbone. This data set isgenerated using a simple steric overlap function where a MHC Class IImolecule is docked with a backbone and an amino-acid side chain isgrafted onto the backbone at the desired position. Each of the rotatablebonds of the side chain is rotated step-wise at set intervals and theresultant positions of the atoms dependent upon that bond noted. Theinteraction of the atom with atoms of side-chains of the binding grooveis noted and positions are either accepted or rejected according to thefollowing criteria: The sum total of the overlap of all atoms so farpositioned must not exceed a pre-determined value.

Thus the stringency of the conformational search is a function of theinterval used in the step-wise rotation of the bond and thepre-determined limit for the total overlap. This latter value can besmall if it is known that a particular pocket is rigid, however thestringency can be relaxed if the positions of pocket side-chains areknown to be relatively flexible. Thus allowances can be made to imitatevariations in flexibility within pockets of the binding groove. Thisconformational search is then repeated for every amino-acid at everyposition of each backbone when docked with each of the MHC Class IImolecules to create the exhaustive database of side-chain conformations.

A suitable mathematical expression is used to estimate the energy ofbinding between models of MHC Class II molecules in conjunction withpeptide ligand conformations which have to be empirically derived byscanning the large database of backbone/side-chain conformationsdescribed above. Thus a protein is scanned for potential T-cell epitopesby subjecting each possible peptide of length varying between 9 and 20amino-acids (although the length is kept constant for each scan) to thefollowing computations: An MHC Class II molecule is selected togetherwith a peptide backbone allowed for that molecule and the side-chainscorresponding to the desired peptide sequence are grafted on. Atomidentity and interatomic distance data relating to a particularside-chain at a particular position on the backbone are collected foreach allowed conformation of that amino-acid (obtained from the databasedescribed above). This is repeated for each side-chain along thebackbone and peptide scores derived using a scoring function. The bestscore for that backbone is retained and the process repeated for eachallowed backbone for the selected model. The scores from all allowedbackbones are compared and the highest score is deemed to be the peptidescore for the desired peptide in that MHC Class II model. This processis then repeated for each model with every possible peptide derived fromthe protein being scanned, and the scores for peptides versus models aredisplayed.

In the context of the present invention, each ligand presented for thebinding affinity calculation is an amino-acid segment selected from apeptide or protein as discussed above. Thus, the ligand is a selectedstretch of amino acids about 9 to 20 amino acids in length derived froma peptide, polypeptide or protein of known sequence. The terms “aminoacids” and “residues” are hereinafter regarded as equivalent terms. Theligand, in the form of the consecutive amino acids of the peptide to beexamined grafted onto a backbone from the backbone library, ispositioned in the binding cleft of an MHC Class II molecule from the MHCClass II molecule model library via the coordinates of the C″-α atoms ofthe peptide backbone and an allowed conformation for each side-chain isselected from the database of allowed conformations. The relevant atomidentities and interatomic distances are also retrieved from thisdatabase and used to calculate the peptide binding score. Ligands with ahigh binding affinity for the MHC Class II binding pocket are flagged ascandidates for site-directed mutagenesis. Amino-acid substitutions aremade in the flagged ligand (and hence in the protein of interest) whichis then retested using the scoring function in order to determinechanges which reduce the binding affinity below a predeterminedthreshold value. These changes can then be incorporated into the proteinof interest to remove T-cell epitopes. Binding between the peptideligand and the binding groove of MHC Class II molecules involvesnon-covalent interactions including, but not limited to: hydrogen bonds,electrostatic interactions, hydrophobic (lipophilic) interactions andVan der Walls interactions. These are included in the peptide scoringfunction as described in detail below. It should be understood that ahydrogen bond is a non-covalent bond which can be formed between polaror charged groups and consists of a hydrogen atom shared by two otheratoms. The hydrogen of the hydrogen donor has a positive charge wherethe hydrogen acceptor has a partial negative charge. For the purposes ofpeptide/protein interactions, hydrogen bond donors may be eithernitrogens with hydrogen attached or hydrogens attached to oxygen ornitrogen. Hydrogen bond acceptor atoms may be oxygens not attached tohydrogen, nitrogens with no hydrogens attached and one or twoconnections, or sulphurs with only one connection. Certain atoms, suchas oxygens attached to hydrogens or imine nitrogens (e.g. C═NH) may beboth hydrogen acceptors or donors. Hydrogen bond energies range from 3to 7 Kcal/mol and are much stronger than Van der Waal's bonds, butweaker than covalent bonds. Hydrogen bonds are also highly directionaland are at their strongest when the donor atom, hydrogen atom andacceptor atom are co-linear. Electrostatic bonds are formed betweenoppositely charged ion pairs and the strength of the interaction isinversely proportional to the square of the distance between the atomsaccording to Coulomb's law. The optimal distance between ion pairs isabout 2.8 Å. In protein/peptide interactions, electrostatic bonds may beformed between arginine, histidine or lysine and aspartate or glutamate.The strength of the bond will depend upon the pKa of the ionizing groupand the dielectric constant of the medium although they areapproximately similar in strength to hydrogen bonds.

Lipophilic interactions are favorable hydrophobic-hydrophobic contactsthat occur between he protein and peptide ligand. Usually, these willoccur between hydrophobic amino acid side chains of the peptide buriedwithin the pockets of the binding groove such that they are not exposedto solvent. Exposure of the hydrophobic residues to solvent is highlyunfavorable since the surrounding solvent molecules are forced tohydrogen bond with each other forming cage-like clathrate structures.The resultant decrease in entropy is highly unfavorable. Lipophilicatoms may be sulphurs which are neither polar nor hydrogen acceptors andcarbon atoms which are not polar.

Van der Waal's bonds are non-specific forces found between atoms whichare 3–4 Å apart. They are weaker and less specific than hydrogen andelectrostatic bonds. The distribution of electronic charge around anatom changes with time and, at any instant, the charge distribution isnot symmetric. This transient asymmetry in electronic charge induces asimilar asymmetry in neighboring atoms. The resultant attractive forcesbetween atoms reaches a maximum at the Van der Waal's contact distancebut diminishes very rapidly at about 1 Å to about 2 Å. Conversely, asatoms become separated by less than the contact distance, increasinglystrong repulsive forces become dominant as the outer electron clouds ofthe atoms overlap. Although the attractive forces are relatively weakcompared to electrostatic and hydrogen bonds (about 0.6 Kcal/mol), therepulsive forces in particular may be very important in determiningwhether a peptide ligand may bind successfully to a protein.

In one embodiment, the Böhm scoring function (SCORE1 approach) is usedto estimate the binding constant. (Böhm, H. J., J. Comput Aided Mol.Des., 8(3):243–256 (1994) which is hereby incorporated in its entirety).In another embodiment, the scoring function (SCORE2 approach) is used toestimate the binding affinities as an indicator of a ligand containing aT-cell epitope (Böhm, H. J., J. Comput Aided Mol. Des., 12(4):309–323(1998) which is hereby incorporated in its entirety). However, the Böhmscoring functions as described in the above references are used toestimate the binding affinity of a ligand to a protein where it isalready known that the ligand successfully binds to the protein and theprotein/ligand complex has had its structure solved, the solvedstructure being present in the Protein Data Bank (“PDB”). Therefore, thescoring function has been developed with the benefit of known positivebinding data. In order to allow for discrimination between positive andnegative binders, a repulsion term must be added to the equation. Inaddition, a more satisfactory estimate of binding energy is achieved bycomputing the lipophilic interactions in a pairwise manner rather thanusing the area based energy term of the above Böhm functions. Therefore,in a preferred embodiment, the binding energy is estimated using amodified Böhm scoring function. In the modified Böhm scoring function,the binding energy between protein and ligand (ΔG_(bind)) is estimatedconsidering the following parameters: The reduction of binding energydue to the overall loss of translational and rotational entropy of theligand (ΔG₀); contributions from ideal hydrogen bonds (ΔG_(hb)) where atleast one partner is neutral; contributions from unperturbed ionicinteractions (ΔG_(ionic)); lipophilic interactions between lipophilicligand atoms and lipophilic acceptor atoms (ΔG_(lipo)); the loss ofbinding energy due to the freezing of internal degrees of freedom in theligand, i.e., the freedom of rotation about each C—C bond is reduced(ΔG_(rot)); the energy of the interaction between the protein and ligand(E_(VdW)). Consideration of these terms gives equation 1:(ΔG _(bind))=(ΔG ₀)+(ΔG _(hb) ×N _(hb))+(ΔG _(ionic) ×N _(ionic))+(ΔG_(lipo) ×N _(lipo))+(ΔG _(rot) +N _(rot))+(E _(VdW)).

Where N is the number of qualifying interactions for a specific termand, in one embodiment, ΔG₀, ΔG_(hb), ΔG_(ionic), ΔG_(lipo) and ΔG_(rot)are constants which are given the values: 5.4, −4.7, −4.7, −0.17, and1.4, respectively.

The term N_(hb) is calculated according to equation 2:

$N_{hb} = {\sum\limits_{h - {bonds}}{f\;\left( {{\Delta\; R},\;{\Delta\alpha}} \right) \times {f\left( N_{neighb} \right)} \times f_{pcs}}}$f(ΔR, Δα) is a penalty function which accounts for large deviations ofhydrogen bonds from ideality and is calculated according to equation 3:f(ΔR,Δ−α)=f1(ΔR)×f2(Δα)

-   Where: f1(ΔR)=1 if ΔR<=TOL    -   or =1−(ΔR−TOL)/0.4 if ΔR<=0.4+TOL    -   or =0 if ΔR>0.4+TOL-   And: f2(Δα)=1 if Δα<30°    -   or =1−(Δα−30)/50 if Δα<=80°    -   or =0 if Δα>80°-   TOL is the tolerated deviation in hydrogen bond length=0.25 Å-   ΔR is the deviation of the H—O/N hydrogen bond length from the ideal    value=1.9 Å-   Δα is the deviation of the hydrogen bond angle ∠_(N/O-H . . . O/N)    from its idealized value of 180°-   f(N_(neighb)) distinguishes between concave and convex parts of a    protein surface and therefore assigns greater weight to polar    interactions found in pockets rather than those found at the protein    surface. This function is calculated according to equation 4 below:    f(N _(neighb))=(N _(neighb) /N _(neighb,0))^(α) where α=0.5-   N_(neighb) is the number of non-hydrogen protein atoms that are    closer than 5 Å to any given protein atom.-   N_(neighb,0) is a constant=25-   f_(pcs) is a function which allows for the polar contact surface    area per hydrogen bond and therefore distinguishes between strong    and weak hydrogen bonds and its value is determined according to the    following criteria:-   f_(pcs)=β when A_(polar)/N_(HB)<10 Å²-   or f_(pcs)=1 when A_(polar)/N_(HB)>10 Å²-   A_(polar) is the size of the polar protein-ligand contact surface-   N_(HB) is the number of hydrogen bonds-   β is a constant whose value=1.2

For the implementation of the modified Böhm scoring function, thecontributions from ionic interactions, ΔG_(ionic), are computed in asimilar fashion to those from hydrogen bonds described above since thesame geometry dependency is assumed.

The term N_(lipo) is calculated according to equation 5 below:

$N_{lipo} = {\sum\limits_{1L}{f\left( r_{1L} \right)}}$

-   f(r_(lL)) is calculated for all lipophilic ligand atoms, l, and all    lipophilic protein atoms, L, according to the following criteria:-   f(r_(lL))=1 when r_(lL)<=R1f(r_(lL))=(r_(lL)−R1)/(R2−R1) when    R2<r_(lL)>R1-   f(r_(lL))=0 when r_(lL)>=R2-   Where: R1=r_(l) ^(vdw)+r_(L) ^(vdw)+0.5-   and R2=R1+3.0-   and r_(l) ^(vdw) is the Van der Waal's radius of atom l-   and r_(L) ^(vdw) is the Van der Waal's radius of atom L

The term N_(rot) is the number of rotable bonds of the amino acid sidechain and is taken to be the number of acyclic sp³-sp³ and sp³-sp²bonds. Rotations of terminal —CH₃ or —NH₃ are not taken into account.

The final term, E_(VdW), is calculated according to equation 6 below:E _(VdW)=ε₁ε₂((r ₁ ^(vdw) +r ₂ ^(vdw))¹² /r ¹²−(r ₁ ^(vdw) +r ₂ ^(vdw))⁶/r ⁶), where:

-   ε₁ and ε₂ are constants dependant upon atom identity-   r₁ ^(vdw)+r₂ ^(vdw) are the Van der Waal's atomic radii-   r is the distance between a pair of atoms.

With regard to Equation 6, in one embodiment, the constants ε₁ and ε₂are given the atom values: C: 0.245, N: 0.283, O: 0.316, S: 0.316,respectively (i.e. for atoms of Carbon, Nitrogen, Oxygen and Sulphur,respectively). With regards to equations 5 and 6, the Van der Waal'sradii are given the atom values C: 1.85, N: 1.75, O: 1.60, S: 2.00 Å.

It should be understood that all predetermined values and constantsgiven in the equations above are determined within the constraints ofcurrent understandings of protein ligand interactions with particularregard to the type of computation being undertaken herein. Therefore, itis possible that, as this scoring function is refined further, thesevalues and constants may change hence any suitable numerical value whichgives the desired results in terms of estimating the binding energy of aprotein to a ligand may be used and hence fall within the scope of thepresent invention.

As described above, the scoring function is applied to data extractedfrom the database of side-chain conformations, atom identities, andinteratomic distances. For the purposes of the present description, thenumber of MHC Class II molecules included in this database is 42 modelsplus four solved structures. It should be apparent from the abovedescriptions that the modular nature of the construction of thecomputational method of the present invention means that new models cansimply be added and scanned with the peptide backbone library andside-chain conformational search function to create additional data setswhich can be processed by the peptide scoring function as describedabove. This allows for the repertoire of scanned MHC Class II moleculesto easily be increased, or structures and associated data to be replacedif data are available to create more accurate models of the existingalleles.

The present prediction method can be calibrated against a data setcomprising a large number of peptides whose affinity for various MHCClass II molecules has previously been experimentally determined. Bycomparison of calculated versus experimental data, a cut of value can bedetermined above which it is known that all experimentally determinedT-cell epitopes are correctly predicted.

It should be understood that, although the above scoring function isrelatively simple compared to some sophisticated methodologies that areavailable, the calculations are performed extremely rapidly. It shouldalso be understood that the objective is not to calculate the truebinding energy per se for each peptide docked in the binding groove of aselected MHC Class II protein. The underlying objective is to obtaincomparative binding energy data as an aid to predicting the location ofT-cell epitopes based on the primary structure (i.e. amino acidsequence) of a selected protein. A relatively high binding energy or abinding energy above a selected threshold value would suggest thepresence of a T-cell epitope in the ligand. The ligand may then besubjected to at least one round of amino-acid substitution and thebinding energy recalculated. Due to the rapid nature of thecalculations, these manipulations of the peptide sequence can beperformed interactively within the program's user interface oncost-effectively available computer hardware. Major investment incomputer hardware is thus not required.

It would be apparent to one skilled in the art that other availablesoftware could be used for the same purposes. In particular, moresophisticated software which is capable of docking ligands into proteinbinding-sites may be used in conjunction with energy minimization.Examples of docking software are: DOCK (Kuntz et al., J. Mol. Biol.,161:269–288 (1982)), LUDI (Böhm, H. J., J. Comput Aided Mol. Des.,8:623–632 (1994)) and FLEXX (Rarey M., et al., ISMB, 3:300–308 (1995)).Examples of molecular modeling and manipulation software include: AMBER(Tripos) and CHARMm (molecular Simulations Inc.). The use of thesecomputational methods would severely limit the throughput of the methodof this invention due to the lengths of processing time required to makethe necessary calculations. However, it is feasible that such methodscould be used as a ‘secondary screen’ to obtain more accuratecalculations of binding energy for peptides which are found to be‘positive binders’ via the method of the present invention. Thelimitation of processing time for sophisticated molecular mechanic ormolecular dynamic calculations is one which is defined both by thedesign of the software which makes these calculations and the currenttechnology limitations of computer hardware. It may be anticipated that,in the future, with the writing of more efficient code and thecontinuing increases in speed of computer processors, it may becomefeasible to make such calculations within a more manageable time-frame.Further information on energy functions applied to macromolecules andconsideration of the various interactions that take place within afolded protein structure can be found in: Brooks, B. R., et al., J.Comput. Chem., 4:187–217 (1983) and further information concerninggeneral protein-ligand interactions can be found in: Dauber-Osguthorpeet al., Proteins 4(1):31–47 (1988), which are incorporated herein byreference in their entirety. Useful background information can also befound, for example, in Fasman, G. D., ed., Prediction of ProteinStructure and the Principles of Protein Conformation, Plenum Press, NewYork, ISBN: 0-306 4313-9.

EXAMPLE 2

De-immunized Forms of Fc-Leptin

Leptin is a secreted signaling 146 amino acid residue protein involvedin the homeostatic mechanisms maintaining adipose mass (e.g. WO00/40615, WO 98/28427, WO 96/05309). The protein (and its antagonists)offers significant therapeutic potential for the treatment of diabetes,high blood pressure and cholesterol metabolism.

Fc-leptin is a fusion protein for which the serum half-life isprofoundly improved compared to leptin itself (WO 0040615). However,certain forms of Fc-leptin, such as when the Fc is derived from humanIgG1 or human IgG3, have the potential to show enhanced immunogenicityunder certain circumstances, such as administration by subcutaneousinjection. In a Phase I clinical trial, leptin alone was found to be atleast somewhat immunogenic. The invention discloses sequences identifiedwithin the leptin primary sequence that are potential T-cell epitopes byvirtue of MHC class II binding potential. This disclosure specificallypertains to the human leptin moiety containing about 146 amino acidresidues. Others have provided modified leptin (U.S. Pat. No. 5,900,404;WO96/05309) but these approaches have been directed towards improvementsin the commercial production of leptin, for example improved in vitrostability. Such teachings do not recognize the importance of T-cellepitopes to the immunogenic properties of the protein nor have beenconceived to directly influence said properties in a specific andcontrolled way according to the scheme of the present invention.Specific Fc-leptin forms: Fcγ1-leptin, Fcγ2-leptin, both forms,preferably with linker peptide and optionally modified Fc domain havingreduced affinity to Fc-receptors. Sequences to be modified in leptin areshown below in Table 1. Substitutions leading to elimination of T-cellepitopes of human leptin are shown in Table 2.

An amino acid sequence which is part of the sequence of animmunogenically non-modified human obesity protein (leptin) and has apotential MHC class II binding activity is selected from the followinggroup shown in Table 1, identified according to the method of theinvention.

Table 1. Peptide sequences in human leptin with potential human MHCclass II binding activity

TABLE 1 Peptide sequences in human leptin with potential human MHC classII binding activity VPIQKVQDDTKTL, QKVQDDTKTLIKT, KTLIKTIVTRIND, (SEQ IDNO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) TLIKTIVTRINDI, KTIVTRINDISHT,TIVTRINDISHTQ, (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10)TRINDISHTQSVS, NDISHTQSVSSKQ, QSVSSKQKVTGLD, (SEQ ID NO: 11) (SEQ ID NO:12) (SEQ ID NO: 13) SSKQKVTGLDFIP, QKVTGLDFIPGLH, TGLDFIPGLHPIL, (SEQ IDNO: 14) (SEQ ID NO: 15) (SEQ ID NO: 16) LDFIPGLHPILTL, DFIPGLHPILTLS,PGLHPILTLSKMD, (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ ID NO: 19)GLHPILTLSKMDQ, HPILTLSKMDQTL, PILTLSKMDQTLA, (SEQ ID NO: 20) (SEQ ID NO:21) (SEQ ID NO: 22) LTLSKMDQTLAVY, SKMDQTLAVYQQI, QTLAVYQQILTSM, (SEQ IDNO: 23) (SEQ ID NO: 24) (SEQ ID NO: 25) LAVYQQILTSMPS, AVYQQILTSMPSR,QQILTSMPSRNVI, (SEQ ID NO: 26) (SEQ ID NO: 27) (SEQ ID NO: 28)QILTSMPSRNVIQ, TSMPSRNVIQISN, SRNVIQISNDLEN, (SEQ ID NO: 29) (SEQ ID NO:30) (SEQ ID NO: 31) RNVIQISNDLENL, NVIQISNDLENLR, IQISNDLENLRDL, (SEQ IDNO: 32) (SEQ ID NO: 33) (SEQ ID NO: 34) NDLENLRDLLHVL, LENLRDLLHVLAF,ENLRDLLHVLAFS, (SEQ ID NO: 35) (SEQ ID NO: 36) (SEQ ID NO: 37)RDLLHVLAFSKSC, DLLHVLAFSKSCH, LHVLAFSKSCHLP, (SEQ ID NO: 38) (SEQ ID NO:39) (SEQ ID NO: 40) HVLAFSKSCHLPW, LAFSKSCHLPWAS, CHLPWASGLETLD, (SEQ IDNO: 41) (SEQ ID NO: 42) (SEQ ID NO: 43) SGLETLDSLGGVL, DSLGGVLEASGYS,SLGGVLEASGYST, (SEQ ID NO: 44) (SEQ ID NO: 45) (SEQ ID NO: 46)GGVLEASGYSTEV, SGYSTEVVALSRL, TEVVALSRLQGSL, (SEQ ID NO: 47) (SEQ ID NO:48) (SEQ ID NO: 49) EVVALSRLQGSLQ, VALSRLQGSLQDM, SRLQGSLQDMLWQ, (SEQ IDNO: 50) (SEQ ID NO: 51) (SEQ ID NO: 52) QGSLQDMLWQLDL, GSLQDMLWQLDLS,QDMLWQLDLSPGC (SEQ ID NO: 53) (SEQ ID NO: 54) (SEQ ID NO: 55)

Table 2. Substitutions leading to the elimination of potential T-cellepitopes of human leptin (WT=wild type).

TABLE 2 Substitutions leading to the elimination of potential T-cellepitopes of human leptin (WT =wild type). Residue # WT residueSubstitutions 3 I A C D E G H K N P Q R S T 6 V A C D E G H K N P Q R ST 13 L A C D E G H K N P Q R S T 14 I A C D E G H K N P Q R S T 17 I A CD E G H K N P Q R S T 18 V A C D E G H K N P Q R S T 21 I A C D E G H KN P Q R S T 24 I A C D E G H K N P Q R S T 30 V A C D E G H K N P Q R ST 36 V A C D E G H K N P Q R S T 39 L A C D E G H K N P Q R S T 41 F A CD E G H K N P Q R S T 42 I A C D E G H K N P Q R S T 45 L A C D E G H KN P Q R S T 48 I A C D E G H K N P Q R S T 49 L A C D E G H K N P Q R ST 51 L A C D E G H K N P Q R S T 54 M A C D E G H K N P Q R S T 58 L A CD E G H K N P Q R S T 60 V A C D E G H K N P Q R S T 61 Y A C D E G H KN P Q R S T 64 I A C D E G H K N P Q R S T 65 L A C D E G H K N P Q R ST 68 M A C D E G H K N P Q R S T 73 V A C D E G H K N P Q R S T 74 I A CD E G H K N P Q R S T 76 I A C D E G H K N P Q R S T 80 L A C D E G H KN P Q R S T 83 L A C D E G H K N P Q R S T 86 L A C D E G H K N P Q R ST 87 L A C D E G H K N P Q R S T 89 V A C D E G H K N P Q R S T 90 L A CD E G H K N P Q R S T 92 F A C D E G H K N P Q R S T 98 L A C D E G H KN P Q R S T 100 W A C D E G H K N P Q R S T 104 L A C D E G H K N P Q RS T 107 L A C D E G H K N P Q R S T 110 L A C D E G H K N P Q R S T 113V A C D E G H K N P Q R S T 114 L A C D E G H K N P Q R S T 119 Y A C DE G H K N P Q R S T 123 V A C D E G H K N P Q R S T 124 V A C D E G H KN P Q R S T 126 L A C D E G H K N P Q R S T 129 L A C D E G H K N P Q RS T 133 L A C D E G H K N P Q R S T 136 M A C D E G H K N P Q R S T

Any of the above-cited peptide sequences can be used for modifying byexchanging one or More amino acids to obtain a sequence having a reducedor no immunogenicity.

EXAMPLE 3

De-immunized Forms of Fc-IL-1Ra

The present invention provides for modified forms of an interleukin-1receptor antagonist (IL-1Ra) with one or more T-cell epitopes removed.IL-1 is an important inflammatory and immune modulating cytokine withpleiotropic effects on a variety of tissues but may contribute to thepathology associated with rheumatoid arthritis and other diseasesassociated with local tissue damage. An IL-1 receptor antagonist able toinhibit the action of IL-1 has been purified and the gene cloned[Eisenburg S. P. et al (1990) Nature, 343: 341–346; Carter, D. B. et al(1990) Nature, 344: 633–637]. Others have provided IL-1Ra molecules[e.g. U.S. Pat. No. 5,075,222]. Recombinant forms of this protein havetherapeutic potential in disease settings where the effects of IL-1 aredeleterious. However, there remains a continued need for IL-1Raanalogues with enhanced properties. Desired enhancements includealternative schemes and modalities for the expression and purificationof the said therapeutic, but also and especially, improvements in thebiological properties of the protein. There is a particular need forenhancement of the in vivo characteristics when administered to thehuman subject. In this regard, it is highly desired to provide IL-1Rawith reduced or absent potential to induce an immune response in thehuman subject. Such proteins would expect to display an increasedcirculation time within the human subject and would be of particularbenefit in chronic or recurring disease settings such as is the case fora number of indications for IL-1Ra. The present invention provides formodified forms of IL-1Ra proteins that are expected to display enhancedproperties in vivo. This disclosure specifically pertains a human IL-1Raprotein being of 152 amino acid residues (Eisenburg, S. P. et al (1991)Proc. Natl. Acad. Sci. U.S.A. 88: 5232–5236).

Specific Fc-IL-1Ra forms: Fcγ1-IL-1Ra, Fcγ2-IL-1Ra, both forms,preferably with linker peptide and optionally modified Fc domain havingreduced affinity to Fc-receptors. Peptide sequences in humaninterleukin-1 receptor antagonist (IL-1RA) with potential human MHCclass II binding activity are shown in Table 3.

Table 3. Potential human MHC class II binding sequences.

TABLE 3 Potential human MHC class II binding sequences RKSSKMQAFRIWD,SKMQAFRIWDVNQ, QAFRIWDVNQKTF, (SEQ ID NO: 56) (SEQ ID NO: 57) (SEQ IDNO: 58) FRIWDVNQKTFYL, RIWDVNQKTFYLR, IWDVNQKTFYLRN, (SEQ ID NO: 59)(SEQ ID NO: 60) (SEQ ID NO: 61) WDVNQKTFYLRNN, KTFYLRNNQLVAG,TFYLRNNQLVAGY, (SEQ ID NO: 62) (SEQ ID NO: 63) (SEQ ID NO: 64)FYLRNNQLVAGYL, LRNNQLVAGYLQG, RNNQLVAGYLQGP, (SEQ ID NO: 65) (SEQ ID NO:66) (SEQ ID NO: 67) NQLVAGYLQGPNV, QLVAGYLQGPNVN, LVAGYLQGPNVNL, (SEQ IDNO: 68) (SEQ ID NO: 69) (SEQ ID NO: 70) AGYLQGPNVNLEE, GYLQGPNVNLEEK,PNVNLEEKIDVVP, (SEQ ID NO: 71) (SEQ ID NO: 72) (SEQ ID NO: 73)VNLEEKIDVVPIE, EKIDVVPIEPHAL, IDVVPIEPHALFL, (SEQ ID NO: 74) (SEQ ID NO:75) (SEQ ID NO: 76) DVVPIEPHALFLG, VPIEPHALFLGIH, HALFLGIHGGKMC, (SEQ IDNO: 77) (SEQ ID NO: 78) (SEQ ID NO: 79) ALFLGIHGGKMCL, LFLGIHGGKMCLS,LGIHGGKMCLSCV, (SEQ ID NO: 80) (SEQ ID NO: 81) (SEQ ID NO: 82)GKMCLSCVKSGDE, MCLSCVKSGDETR, SCVKSGDETRLQL, (SEQ ID NO: 83) (SEQ ID NO:84) (SEQ ID NO: 85) ETRLQLEAVNITD, TRLQLEAVNITDL, LQLEAVNITDLSE, (SEQ IDNO: 86) (SEQ ID NO: 87) (SEQ ID NO: 88) EAVNITDLSENRK, VNITDLSENRKQD,TDLSENRKQDKRF, (SEQ ID NO: 89) (SEQ ID NO: 90) (SEQ ID NO: 91)ENRKQDKRFAFIR, KRFAFIRSDSGPT, FAFIRSDSGPTTS, (SEQ ID NO: 92) (SEQ ID NO:93) (SEQ ID NO: 94) AFIRSDSGPTTSF, TSFESAACPGWFL, SFESAACPGWFLC, (SEQ IDNO: 95) (SEQ ID NO: 96) (SEQ ID NO: 97) PGWFLCTAMEADQ, WFLCTAMEADQPV,TAMEADQPVSLTN, (SEQ ID NO: 98) (SEQ ID NO: 99) (SEQ ID NO: 100)QPVSLTNMPDEGV, VSLTNMPDEGVMV, TNMPDEGVMVTKF, (SEQ ID NO: 101) (SEQ IDNO: 102) (SEQ ID NO: 103) PDEGVMVTKFYFQ, EGVMVTKFYFQED, GVMVTKFYFQEDE(SEQ ID NO: 104) (SEQ ID NO: 105) (SEQ ID NO: 106)

Substitutions leading to the elimination of potential T-cell epitopes ofhuman interleukin-1 receptor antagonist (IL-1RA) (WT=wild type) areshown in Table 4.

Table 4. Potential T-cell epitopes of human interleukin-1 receptorantagonist.

TABLE 4 Potential T-cell epitopes of human interleukin-1 antagonist.Residue # WT Residue Substitution 10 M A C D E G H K N P Q R S T 13 F AC D E G H K N P Q R S T 15 I A C D E G H K N P Q R S T 16 W A C D E G HK N P Q R S T 18 V A C D E G H K N P Q R S T 23 F A C D E G H K N P Q RS T 24 Y A C D E G H K N P Q R S T 25 L A C D E G H K N P Q R S T 30 L AC D E G H K N P Q R S T 31 V A C D E G H K N P Q R S T 34 Y A C D E G HK N P Q R S T 35 L A C D E G H K N P Q R S T 40 V A C D E G H K N P Q RS T 42 L A C D E G H K N P Q R S T 46 I A C D E G H K N P Q R S T 48 V AC D E G H K N P Q R S T 49 V A C D E G H K N P Q R S T 51 I A C D E G HK N P Q R S T 56 L A C D E G H K N P Q R S T 57 F A C D E G H K N P Q RS T 58 L A C D E G H K N P Q R S T 60 I A C D E G H K N P Q R S T 65 M AC D E G H K N P Q R S T 67 L A C D E G H K N P Q R S T 70 V A C D E G HK N P Q R S T 78 L A C D E G H K N P Q R S T 80 L A C D E G H K N P Q RS T 83 V A C D E G H K N P Q R S T 85 I A C D E G H K N P Q R S T 88 L AC D E G H K N P Q R S T 98 F A C D E G H K N P Q R S T 100 F A C D E G HK N P Q R S T 101 I A C D E G H K N P Q R S T 119 W A C D E G H K N P QR S T 120 F A C D E G H K N P Q R S T 121 L A C D E G H K N P Q R S T125 M A C D E G H K N P Q R S T 131 V A C D E G H K N P Q R S T 133 L AC D E G H K N P Q R S T 136 M A C D E G H K N P Q R S T 141 V A C D E GH K N P Q R S T 142 M A C D E G H K N P Q R S T

EXAMPLE 4

De-immunized Forms of Fc-BNDF

The present invention provides for modified forms of human brain-derivedneurotrophic factor (BDNF) with one or more T-cell epitopes removed.BDNF is glycoprotein of the nerve growth factor family of proteins. Themature 119 amino acid glycoprotein is processed from a larger pre-cursorto yield a neurotrophic factor that promotes the survival of neuronalcell populations [Jones K. R. & Reichardt, L. F. (1990) Proc. Natl.Acad. Sci U.S.A. 87: 8060–8064]. Others have provided modified BDNFmolecules [U.S. Pat. No. 5,770,577] and approaches towards thecommercial production of recombinant BDNF molecules [U.S. Pat. No.5,986,070]. Such neuronal cells are all located either in the centralnervous system or directly connected to it. Recombinant preparations ofBDNF have enabled the therapeutic potential of the protein to beexplored for the promotion of nerve regeneration and degenerativedisease therapy. Specific Fc-BDNF forms: Fcγ1-BDNF, Fcγ2-BDNF, bothforms, preferably with linker peptide and optionally modified Fc domainhaving reduced affinity to Fc-receptors. Peptide sequences in humanbrain-derived neurotrophic factor (BDNF) with potential human MHC classII binding activity are shown in Table 5.

Table 5. Potential T-cell epitopes of human brain-derived neurotrophicfactor.

TABLE 5 Potential T-cell epitopes of human brain-derived neurotrophicfactor GELSVCDSISEWV, LSVCDSISEWVTA, DSISEWVTAADKK, (SEQ ID NO: 107)(SEQ ID NO: 108) (SEQ ID NO: 109) SEWVTAADKKTAV, EWVTAADKKTAVD,WVTAADKKTAVDM, (SEQ ID NO: 110) (SEQ ID NO: 111) (SEQ ID NO: 112)KTAVDMSGGTVTV, TAVDMSGGTVTVL, VDMSGGTVTVLEK, (SEQ ID NO: 113) (SEQ IDNO: 114) (SEQ ID NO: 115) GTVTVLEKVPVSK, VTVLEKVPVSKGQ, TVLEKVPVSKGQL,(SEQ ID NO: 116) (SEQ ID NO: 117) (SEQ ID NO: 118) EKVPVSKGQLKQY,VPVSKGQLKQYFY, GQLKQYFYETKCN, (SEQ ID NO: 119) (SEQ ID NO: 120) (SEQ IDNO: 121) KQYFYETKCNPMG, QYFYETKCNPMGY, YFYETKCNPMGYT, (SEQ ID NO: 122)(SEQ ID NO: 123) (SEQ ID NO: 124) NPMGYTKEGCRGI, MGYTKEGCRGIDK,RGIDKRHWNSQCR, (SEQ ID NO: 125) (SEQ ID NO: 126) (SEQ ID NO: 127)RHWNSQCRTTQSY, HWNSQCRTTQSYV, QSYVRALTMDSKK, (SEQ ID NO: 128) (SEQ IDNO: 129) (SEQ ID NO: 130) SYVRALTMDSKKR, RALTMDSKKRIGW, LTMDSKKRIGWRF,(SEQ ID NO: 131) (SEQ ID NO: 132) (SEQ ID NO: 133) KRIGWRFIRIDTS,IGWRFIRIDTSCV, GWRFIRIDTSCVC, (SEQ ID NO: 134) (SEQ ID NO: 135) (SEQ IDNO: 136) WRFIRIDTSCVCT, RFIRIDTSCVCTL, IRIDTSCVCTLTI, (SEQ ID NO: 137)(SEQ ID NO: 138) (SEQ ID NO: 139) IDTSCVCTLTIKR (SEQ ID NO: 140)

Substitutions leading to the elimination of potential T-cell epitopes ofhuman brain-derived neurotrophic factor (BDNF) (WT=wild type) are shownin Table 6.

Table 6. Substitutions leading to elimination of potential T-cellepitopes of human brain-derived neurotrophic factor.

TABLE 6 Substitutions leading to elimination of potential T-cellepitopes of human brain-derived neurotrophic factor. Residue # WTResidue Substitution 10 L A C D E G H K N P Q R S T 16 I A C D E G H K NP Q R S T 20 V A C D E G H K N P Q R S T 29 V A C D E G H K N P Q R S T31 M A C D E G H K N P Q R S T 36 V A C D E G H K N P Q R S T 38 V A C DE G H K N P Q R S T 39 L A C D E G H K N P Q R S T 42 V A C D E G H K NP Q R S T 44 V A C D E G H K N P Q R S T 49 L A C D E G H K N P Q R S T52 Y A C D E G H K N P Q R S T 53 F A C D E G H K N P Q R S T 54 Y A C DE G H K N P Q R S T 61 M A C D E G H K N P Q R S T 63 Y A C D E G H K NP Q R S T 71 I A C D E G H K N P Q R S T 76 W A C D E G H K N P Q R S T86 Y A C D E G H K N P Q R S T 87 V A C D E G H K N P Q R S T 90 L A C DE G H K N P Q R S T 92 M A C D E G H K N P Q R S T 98 I A C D E G H K NP Q R S T 100 W A C D E G H K N P Q R S T 102 F A C D E G H K N P Q R ST 103 I A C D E G H K N P Q R S T 105 I A C D E G H K N P Q R S T

EXAMPLE 5

De-immunized Forms of Fc-EPO

The present invention provides for modified forms of humanerythropioetin (EPO) with one or more T-cell epitopes removed. EPO is a165 amino acid residues glycoprotein hormone involved in the maturationof erythroid progenitor cells into erythrocytes. Naturally occurring EPOis produced by the liver during fetal life and by the kidney of adultsand circulates in the blood to stimulate production of red blood cellsin bone marrow. Anaemia is almost invariably a consequence of renalfailure due to decreased production of EPO from the kidney. RecombinantEPO is used as an effective treatment of anaemia resulting from chronicrenal failure.

Recombinant EPO (expressed in mammalian cells) having the amino acidsequence 1–165 of human erythropoietin [Jacobs, K. et al (1985) Nature,313: 806–810; Lin, F.-K. et al (1985) Proc. Natl. Acad. Sci. U.S.A.82:7580–7585] contains three N-linked and one O-linked oligosaccharidechains each containing terminal sialic acid residues. The latter aresignificant in enabling EPO to evade rapid clearance from thecirculation by the hepatic asialoglycoprotein binding protein.

Non-de-immunized Fc-EPO is known e.g. from WO 99/58662, WO 99/02709.Specific Fc-EPO forms are: Fcγ1-EPO, Fcγ2-EPO, both forms, preferablyhaving a linker peptide and optionally a modified Fc domain havingreduced affinity to Fc-receptors. The EPO may be glycosylated, partiallyglycosylated or have a modified glycosylation pattern. Peptide sequencesin human erythropoietin (EPO) with potential human MHC class II bindingactivity are shown in Table 7.

Table 7. Potential T-cell epitopes of human erythropoietin.

TABLE 7 Potential T-cell epitopes of human erythropoietin PRLICDSRVLERY,RLICDSRVLERYL, ICDSRVLERYLLE, (SEQ ID NO: 141) (SEQ ID NO: 142) (SEQ IDNO: 143) CDSRVLERYLLEA, SRVLERYLLEAKE, RVLERYLLEAKEA, (SEQ ID NO: 144)(SEQ ID NO: 145) (SEQ ID NO: 146) LERYLLEAKEAEN, ERYLLEAKEAENI,RYLLEAKEAENIT, (SEQ ID NO: 147) (SEQ ID NO: 148) (SEQ ID NO: 149)YLLEAKEAENITT, LEAKEAENITTGC, KEAENITTGCAEH, (SEQ ID NO: 150) (SEQ IDNO: 151) (SEQ ID NO: 152) ENITTGCAEHCSL CSLNENITPDTK NENITVPDTKVNF (SEQID NO: 153) (SEQ ID NO: 154) (SEQ ID NO: 155) ENITVPDTKVNFY,NITVPDTKVNFYA, ITVPDTKVNFYAW, (SEQ ID NO: 156) (SEQ ID NO: 157) (SEQ IDNO: 158) TKVNFYAWKRMEV, VNFYAWKRMEVGQ, NFYAWKRMEVGQQ, (SEQ ID NO: 159)(SEQ ID NO: 160) (SEQ ID NO: 161) YAWKRMEVGQQAV, KRMEVGQQAVEVW,RMEVGQQAVEVWQ, (SEQ ID NO: 162) (SEQ ID NO: 163) (SEQ ID NO: 164)MEVGQQAVEVWQG, QAVEVWQGLALLS, AVEVWQGLALLSE, (SEQ ID NO: 165) (SEQ IDNO: 166) (SEQ ID NO: 167) VEVWQGLALLSEA, EVWQGLALLSEAV, VWQGLALLSEAVL,(SEQ ID NO: 168) (SEQ ID NO: 169) (SEQ ID NO: 170) WQGLALLSEAVLR,QGLALLSEAVLRG, LALLSEAVLRGQA, (SEQ ID NO: 171) (SEQ ID NO: 172) (SEQ IDNO: 173) ALLSEAVLRGQAL, LSEAVLRGQALLV, SEAVLRGQALLVN, (SEQ ID NO: 174)(SEQ ID NO: 175) (SEQ ID NO: 176) EAVLRGQALLVNS, AVLRGQALLVNSS,QALLVNSSQPWEP, (SEQ ID NO: 177) (SEQ ID NO: 178) (SEQ ID NO: 179)ALLVNSSQPWEPL, LLVNSSQPWEPLQ, QPWEPLQLHVDKA, (SEQ ID NO: 180) (SEQ IDNO: 181) (SEQ ID NO: 182) EPLQLHVDKAVSG, LQLHVDKAVSGLR, LHVDKAVSGLRSL,(SEQ ID NO: 183) (SEQ ID NO: 184) (SEQ ID NO: 185) KAVSGLRSLTTLL,SGLRSLTTLLRAL, RSLTTLLRALGAQ, (SEQ ID NO: 186) (SEQ ID NO: 187) (SEQ IDNO: 188) SLTTLLRALGAQK, TTLLRALGAQKEA, TLLRALGAQKEAI, (SEQ ID NO: 189)(SEQ ID NO: 190) (SEQ ID NO: 191) RALGAQKEAISPP, AQKEATSPPDAAS,EAISPPDAASAAP, (SEQ ID NO: 192) (SEQ ID NO: 193) (SEQ ID NO: 194)SPPDAASAAPLRT, ASAAPLRTITADT, APLRTITADTFRK, (SEQ ID NO: 195) (SEQ IDNO: 196) (SEQ ID NO: 197) RTITADTFRKLFR, TITADTFRKLFRV, DTFRKLFRVYSNF,(SEQ ID NO: 198) (SEQ ID NO: 199) (SEQ ID NO: 200) RKLFRVYSNFLRG,KLFRVYSNFLRGK, FRVYSNFLRGKLK, (SEQ ID NO: 201) (SEQ ID NO: 202) (SEQ IDNO: 203) RVYSNFLRGKLKL, YSNFLRGKLKLYT, SNFLRGKLKLYTG, (SEQ ID NO: 204)(SEQ ID NO: 205) (SEQ ID NO: 206) NFLRGKLKLYTGE, RGKLKLYTGEACR,GKLKLYTGEACRT, (SEQ ID NO: 207) (SEQ ID NO: 208) (SEQ ID NO: 209)LKLYTGEACRTGD, KLYTGEACRTGDR (SEQ ID NO: 210) (SEQ ID NO: 211)

Substitutions leading to the elimination of potential T-cell epitopes ofhuman erythropoietin (EPO) (WT=wild type) are shown in Table 8.

Table 8. Substitutions leading to elimination of potential T-cellepitopes of human EPO.

TABLE 8 Substitutions leading to elimination of potential T-cellepitopes of human EPO. Residue # WT residue Substitutions 5 L A C D E GH K N P Q R S T 6 I A C D E G H K N P Q R S T 11 V A C D E G H K N P Q RS T 12 L A C D E G H K N P Q R S T 15 Y A C D E G H K N P Q R S T 16 L AC D E G H K N P Q R S T 17 L A C D E G H K N P Q R S T 25 I A C D E G HK N P Q R S T 35 L A C D E G H K N P Q R S T 39 I A C D E G H K N P Q RS T 41 V A C D E G H K N P Q R S T 46 V A C D E G H K N P Q R S T 48 F AC D E G H K N P Q R S T 49 Y A C D E G H K N P Q R S T 51 W A C D E G HK N P Q R S T 54 M A C D E G H K N P Q R S T 56 V A C D E G H K N P Q RS T 61 V A C D E G H K N P Q R S T 63 V A C D E G H K N P Q R S T 64 W AC D E G H K N P Q R S T 67 L A C D E G H K N P Q R S T 69 L A C D E G HK N P Q R S T 70 L A C D E G H K N P Q R S T 74 V A C D E G H K N P Q RS T 75 L A C D E G H K N P Q R S T 80 L A C D E G H K N P Q R S T 81 L AC D E G H K N P Q R S T 82 N A C D E G H K N P Q R S T 88 W A C D E G HK N P Q R S T 91 L A C D E G H K N P Q R S T 93 L A C D E G H K N P Q RS T 95 V A C D E G H K N P Q R S T 99 V A C D E G H K N P Q R S T 102 LA C D E G H K N P Q R S T 105 L A C D E G H K N P Q R S T 108 L A C D EG H K N P Q R S T 109 L A C D E G H K N P Q R S T 112 L A C D E G H K NP Q R S T 119 I A C D E G H K N P Q R S T 130 L A C D E G H K N P Q R ST 133 I A C D E G H K N P Q R S T 138 F A C D E G H K N P Q R S T 141 LA C D E G H K N P Q R S T 142 F A C D E G H K N P Q R S T 144 V A C D EG H K N P Q R S T 145 Y A C D E G H K N P Q R S T 148 F A C D E G H K NP Q R S T 149 L A C D E G H K N P Q R S T 153 L A C D E G H K N P Q R ST 155 L A C D E G H K N P Q R S T 156 Y A C D E G H K N P Q R S T

EXAMPLE 6

De-immunized Forms of G-CSF

The present invention provides for modified forms of human granulocytecolony stimulating factor (G-CSF) with one or more T-cell epitopesremoved. G-CSF is an important haemopoietic cytokine currently used intreatment of indications where an increase in blood neutrophils willprovide benefits. These include cancer therapy, various infectiousdiseases and related conditions such as sepsis. G-CSF is also usedalone, or in combination with other compounds and cytokines in the exvivo expansion of haemopoeitic cells for bone marrow transplantation.

Two forms of human G-CSF are commonly recognized for this cytokine. Oneis a protein of 177 amino acids, the other a protein of 174 amino acids[Nagata et al. (1986), EMBO J. 5: 575–581], the 174 amino acid form hasbeen found to have the greatest specific in vivo biological activity.Recombinant DNA techniques have enabled the production of commercialscale quantities of G-CSF exploiting both eukaryotic and prokaryotichost cell expression systems. This disclosure specifically pertains toboth recognized forms of the human G-CSF protein being the 177 aminoacid species and the 174 amino acid species.

Other polypeptide analogues and peptide fragments of G-CSF have beenpreviously disclosed, including forms modified by site-specific aminoacid substitutions and or by modification by chemical adducts. Thus U.S.Pat. No. 4,810,643 discloses analogues with the particular Cys residuesreplaced with another amino acid, and G-CSF with an Ala residue in thefirst (N-terminal) position. EP 0 335 423 discloses the modification ofat least one amino group in a polypeptide having G-CSF activity. EP 0272 703 discloses G-CSF derivatives having amino acid substituted ordeleted near the N-terminus. EP 0 459 630 discloses G-CSF derivatives inwhich Cys 17 and Asp 27 are replaced by Ser residues. EP 0 243 153discloses G-CSF modified by inactivating at least one yeast KEX2protease processing site for increased yield in recombinant productionand U.S. Pat. No. 4,904,584 discloses lysine altered proteins. WO90/12874 discloses further Cys altered variants and Australian patentdocument AU 10948/92 discloses the addition of amino acids to eitherterminus of a G-CSF molecule for the purpose of aiding in the folding ofthe molecule after prokaryotic expression. A further Australiandocument; AU 76380/91, discloses G-CSF variants at positions 50–56 ofthe G-CSF 174 amino acid form, and positions 53–59 of the 177 amino acidform. Additional changes at particular His residues were also disclosed.

Non-deimmunized Fc-G-CSF is known e.g. from WO 99/58662. SpecificFc-G-CSF forms: Fcγ1-G-CSF, Fcγ2-G-CSF, both forms, preferably withlinker peptide and optionally modified Fc domain having reduced affinityto Fc-receptors.

Peptide sequences in human granulocyte colony stimulating factor (G-CSF)with potential human MHC class II binding activity are shown in Table 9.

Table 9. Potential T-cell epitopes of human G-CSF.

TABLE 9 Potential T-cell epitopes of human G-CSF TPLGPASSLPQSF,SSLPQSFLLKCLE, QSFLLKCLEQVRK, (SEQ ID NO: 212) (SEQ ID NO: 213) (SEQ IDNO: 214) SFLLKCLEQVRKI, FLLKCLEQVRKIQ, KCLEQVRKIQGDG, (SEQ ID NO: 215)(SEQ ID NO: 216) (SEQ ID NO: 217) EQVRKIQGDGAAL, RKIQGDGAALQEK,AALQEKLVSECAT, (SEQ ID NO: 218) (SEQ ID NO: 219) (SEQ ID NO: 220)EKLVSECATYKLC, KLVSECATYKLCH, AALQEKLCATYKL, (SEQ ID NO: 221) (SEQ IDNO: 222) (SEQ ID NO: 223) EKLCATYKLCHPE, ATYKLCHPEELVL, YKLCHPEELVLLG,(SEQ ID NO: 224) (SEQ ID NO: 225) (SEQ ID NO: 226) EELVLLGHSLGIP,ELVLLGHSLGIPW, HSLGIPWAPLSSC, (SEQ ID NO: 227) (SEQ ID NO: 228) (SEQ IDNO: 229) IPWAPLSSCPSQA, APLSSCPSQALQL, QALQLAGCLSQLH, (SEQ ID NO: 230)(SEQ ID NO: 231) (SEQ ID NO: 232) GCLSQLHSGLFLY, SQLHSGLFLYQGL,SGLFLYQGLLQAL, (SEQ ID NO: 233) (SEQ ID NO: 234) (SEQ ID NO: 235)GLFLYQGLLQALE, LFLYQGLLQALEG, FLYQGLLQALEGI, (SEQ ID NO: 236) (SEQ IDNO: 237) (SEQ ID NO: 238) QGLLQALEGISPE, GLLQALEGISPEL, QALEGISPELGPT,(SEQ ID NO: 239) (SEQ ID NO: 240) (SEQ ID NO: 241) EGISPELGPTLDT,PTLDTLQLDVADF, DTLQLDVADFATT, (SEQ ID NO: 242) (SEQ ID NO: 243) (SEQ IDNO: 244) LQLDVADFATTIW, LDVADFATTIWQQ, TTIWQQMEELGMA, (SEQ ID NO: 245)(SEQ ID NO: 246) (SEQ ID NO: 247) TIWQQMEELGMAP, QQMEELGMAPALQ,EELGMAPALQPTQ, (SEQ ID NO: 248) (SEQ ID NO: 249) (SEQ ID NO: 250)LGMAPALQPTQGA, PALQPTQGAMPAF, GAMPAFASAFQRR, (SEQ ID NO: 251) (SEQ IDNO: 252) (SEQ ID NO: 253) PAFASAFQRRAGG, SAFQRRAGGVLVA, GGVLVASHLQSFL,(SEQ ID NO: 254) (SEQ ID NO: 255) (SEQ ID NO: 256) GVLVASHLQSFLE,VLVASHLQSFLEV, SHLQSFLEVSYRV, (SEQ ID NO: 257) (SEQ ID NO: 258) (SEQ IDNO: 259) QSFLEVSYRVLRH, SFLEVSYRVLRHL, LEVSYRVLRHLAQ (SEQ ID NO: 260)(SEQ ID NO: 261) (SEQ ID NO: 262)

Substitutions leading to the elimination of potential T-cell epitopes ofhuman granulocyte colony stimulating factor (G-CSF) (WT=wild type) areshown in Table 10.

Table 10. Substitutions leading to elimination of potential T-cellepitopes of human G-CSF.

TABLE 10 Substitutions leading to elimination of potential T-cellepitopes of human G-CSF. Residue # WT Residue Substitution 3 L A C D E GH K N P Q R S T 9 L A C D E G H K N P Q R S T 14 L A C D E G H K N P Q RS T 15 L A C D E G H K N P Q R S T 18 L A C D E G H K N P Q R S T 21 V AC D E G H K N P Q R S T 24 I A C D E G H K N P Q R S T 31 L A C D E G HK N P Q R S T 35 L A C D E G H K N P Q R S T 39 Y A C D E G H K N P Q RS T 41 L A C D E G H K N P Q R S T 47 L A C D E G H K N P Q R S T 48 V AC D E G H K N P Q R S T 49 L A C D E G H K N P Q R S T 50 L A C D E G HK N P Q R S T 54 L A C D E G H K N P Q R S T 56 I A C D E G H K N P Q RS T 58 W A C D E G H K N P Q R S T 61 L A C D E G H K N P Q R S T 69 L AC D E G H K N P Q R S T 71 L A C D E G H K N P Q R S T 75 L A C D E G HK N P Q R S T 78 L A C D E G H K N P Q R S T 82 L A C D E G H K N P Q RS T 83 F A C D E G H K N P Q R S T 84 L A C D E G H K N P Q R S T 85 Y AC D E G H K N P Q R S T 88 L A C D E G H K N P Q R S T 89 L A C D E G HK N P Q R S T 92 L A C D E G H K N P Q R S T 95 I A C D E G H K N P Q RS T 99 L A C D E G H K N P Q R S T 103 L A C D E G H K N P Q R S T 106 LA C D E G H K N P Q R S T 108 L A C D E G H K N P Q R S T 110 V A C D EG H K N P Q R S T 113 F A C D E G H K N P Q R S T 117 I A C D E G H K NP Q R S T 118 W A C D E G H K N P Q R S T 121 M A C D E G H K N P Q R ST 124 L A C D E G H K N P Q R S T 130 L A C D E G H K N P Q R S T 137 MA C D E G H K N P Q R S T 140 F A C D E G H K N P Q R S T 144 F A C D EG H K N P Q R S T 151 V A C D E G H K N P Q R S T 152 L A C D E G H K NP Q R S T 153 V A C D E G H K N P Q R S T 157 L A C D E G H K N P Q R ST 160 F A C D E G H K N P Q R S T 161 L A C D E G H K N P Q R S T 163 VA C D E G H K N P Q R S T

EXAMPLE 7

De-immunized Forms of KGF

The present invention provides for modified forms of human keratinocytegrowth factor (KGF) with one or more T-cell epitopes removed. KGF is amember of the fibroblast growth factor (FGF)/heparin-binding growthfactor family of proteins. It is a secreted glycoprotein expressedpredominantly in the lung, promoting wound healing by stimulating thegrowth of keratinocytes and other epithelial cells [Finch et al (1989),Science 24: 752–755; Rubin et al (1989), Proc. Natl. Acad. Sci. U.S.A.86: 802–806]. The mature (processed) form of the glycoprotein comprises163 amino acid residues and may be isolated from conditioned mediafollowing culture of particular cell lines [Rubin et al, (1989) ibid.],or produced using recombinant techniques [Ron et al (1993) J. Biol.Chem. 268: 2984–2988]. The protein is of therapeutic value for thestimulation of epithelial cell growth in a number of significant diseaseand injury repair settings. This disclosure specifically pertains thehuman KGF protein being the mature (processed) form of 163 amino acidresidues. Others have also provided KGF molecules [e.g. U.S. Pat. No.6,008,328; WO90/08771;] including modified KGF [Ron et al (1993) ibid;WO9501434] but

Specific Fc-KGF forms: Fcγ1-KGF, Fcγ2-KGF, both forms, preferably withlinker peptide and optionally modified Fc domain having reduced affinityto Fc-receptors.

Peptide sequences in human keratinocyte growth factor (KGF) withpotential human MHC class II binding activity are shown in Table 11.

Table 11. Potential T-cell epitopes of human KGF.

TABLE 11 Potential T-cell epitopes of hman KGF NDMTPEQMATNVN,DMTPEQMATNVNC, EQMATNVNCSSPE, (SEQ ID NO: 263) (SEQ ID NO: 264) (SEQ IDNO: 265) TNVNCSSPERHTR, RSYDYMEGGDIRV, YDYMEGGDIRVRR, (SEQ ID NO: 266)(SEQ ID NO: 267) (SEQ ID NO: 268) DYMEGGDIRVRRL, GDIRVRRLFCRTQ,IRVRRLFCRTQWY, (SEQ ID NO: 269) (SEQ ID NO: 270) (SEQ ID NO: 271)RRLFCRTQWYLRI, RLFCRTQWYLRID, TQWYLRIDKRGKV, (SEQ ID NO: 272) (SEQ IDNO: 273) (SEQ ID NO: 274) QWYLRIDKRGKVK, WYLRIDKRGKVKG, LRIDKRGKVKGTQ,(SEQ ID NO: 275) (SEQ ID NO: 276) (SEQ ID NO: 277) GKVKGTQEMKNNY,QEMKNNYNIMEIR, NNYNIMEIRTVAV, (SEQ ID NO: 278) (SEQ ID NO: 279) (SEQ IDNO: 280) YNIMEIRTVAVGI, NIMEIRTVAVGIV, MEIRTVAVGIVAI, (SEQ ID NO: 281)(SEQ ID NO: 282) (SEQ ID NO: 283) RTVAVGIVAIKGV, VAVGIVAIKGVES,VGIVAIKGVESEF, (SEQ ID NO: 284) (SEQ ID NO: 285) (SEQ ID NO: 286)VAIKGVESEFYLA, KGVESEFYLAMNK, SEFYLAMNKEGKL, (SEQ ID NO: 287) (SEQ IDNO: 288) (SEQ ID NO: 289) EFYLAMNKEGKLY, FYLAMNKEGKLYA, LAMNKEGKLYAKK,(SEQ ID NO: 290) (SEQ ID NO: 291) (SEQ ID NO: 292) GKLYAKKECNEDC,KLYAKKECNEDCN, CNFKELILENHYN, (SEQ ID NO: 293) (SEQ ID NO: 294) (SEQ IDNO: 295) KELILENHYNTYA, ELILENHYNTYAS, LILENHYNTYASA, (SEQ ID NO: 296)(SEQ ID NO: 297) (SEQ ID NO: 298) NHYNTYASAKWTH, NTYASAKWTHNGG,AKWTHNGGEMFVA, (SEQ ID NO: 299) (SEQ ID NO: 300) (SEQ ID NO: 301)GEMFVALNQKGIP, EMFVALNQKGIPV, FVALNQKGIPVRG, (SEQ ID NO: 302) (SEQ IDNO: 303) (SEQ ID NO: 304) VALNQKGIPVRGK, KGIPVRGKKTKKE, IPVRGKKTKKEQK,(SEQ ID NO: 305) (SEQ ID NO: 306) (SEQ ID NO: 307) KTKKEQKTAHFLP (SEQ IDNO: 308)

Substitutions leading to the elimination of potential T-cell epitopes ofhuman keratinocyte growth factor (KGF) (WT=wild type) are shown in Table12.

Table 12. Substitutions leading to elimination of potential T-cellepitopes of human KGF.

TABLE 12 Substitutions leading to elimination of potential T-cellepitopes of human KGF. Residue # WT residue Substitution 5 M A C D E G HK N P Q R S T 10 M A C D E G H K N P Q R S T 14 V A C D E G H K N P Q RS T 26 Y A C D E G H K N P Q R S T 28 Y A C D E G H K N P Q R S T 29 M AC D E G H K N P Q R S T 34 I A C D E G H K N P Q R S T 36 V A C D E G HK N P Q R S T 39 L A C D E G H K N P Q R S T 40 F A C D E G H K N P Q RS T 45 W A C D E G H K N P Q R S T 46 Y A C D E G H K N P Q R S T 47 L AC D E G H K N P Q R S T 49 I A C D E G H K N P Q R S T 55 V A C D E G HK N P Q R S T 61 M A C D E G H K N P Q R S T 65 Y A C D E G H K N P Q RS T 67 I A C D E G H K N P Q R S T 68 M A C D E G H K N P Q R S T 70 I AC D E G H K N P Q R S T 73 V A C D E G H K N P Q R S T 75 V A C D E G HK N P Q R S T 77 I A C D E G H K N P Q R S T 78 V A C D E G H K N P Q RS T 80 I A C D E G H K N P Q R S T 83 V A C D E G H K N P Q R S T 87 F AC D E G H K N P Q R S T 88 Y A C D E G H K N P Q R S T 89 L A C D E G HK N P Q R S T 91 M A C D E G H K N P Q R S T 97 L A C D E G H K N P Q RS T 98 Y A C D E G H K N P Q R S T 109 F A C D E G H K N P Q R S T 112 LA C D E G H K N P Q R S T 113 I A C D E G H K N P Q R S T 114 L A C D EG H K N P Q R S T 118 Y A C D E G H K N P Q R S T 121 Y A C D E G H K NP Q R S T 126 W A C D E G H K N P Q R S T 133 M A C D E G H K N P Q R ST 134 F A C D E G H K N P Q R S T 135 V A C D E G H K N P Q R S T 137 LA C D E G H K N P Q R S T 142 I A C D E G H K N P Q R S T 144 V A C D EG H K N P Q R S T

EXAMPLE 8

De-immunized sTNF-R(1) and sTNF Inhibitor within Corresponding FcFusions

TNF-R(I) and Fc-sTNF Inhibitor are fusion proteins in which the serumhalf-life is extended compared to sTNF-R(I) and sTNF Inhibitor itself.However, certain forms of Fc-TNF-RI, such as when the Fc is derived fromhuman IgG1 or human IgG3, have the potential to show enhancedimmunogenicity under certain circumstances, such as administration bysubcutaneous injection. The present invention provides for modifiedforms of a soluble tumor necrosis factor receptor type I (sTNF-RI) withone or more T-cell epitopes removed. The sTNF-RI (soluble tumor necrosisfactor receptor type I) is a derivative of the human tumor necrosisfactor receptor described previously [Gray, P. W. et al (1990) Proc.Nat. Acad. Sci. U.S.A. 87: 7380–7384; Loetschere, H. et al, (1990) Cell61: 351–359; Schall, T. J. et al (1990) Cell 61: 361–370], comprisingthe extracellular domain of the intact receptor and exhibiting anapproximate molecular weight of 30 KDa. Additional soluble TNFinhibitors and in particular a 40 KDa form are also known [U.S. Pat. No.6,143,866]. The soluble forms are able to bind tumor necrosis factoralpha with high affinity and inhibit the cytotoxic activity of thecytokine in vitro. Recombinant preparations of sTNF-RI are ofsignificant therapeutic value for the treatment of diseases where anexcess level of tumor necrosis factor is causing a pathogenic effect.Indications such as cachexia, sepsis and autoimmune disorders including,and in particular, rheumatoid arthritis and others may be targeted bysuch therapeutic preparations of sTNF-RI. Others including Brewer etal., U.S. Pat. No. 6,143,866, have provided modified sTNF-RI molecules

Peptide sequences in a human 30KDa sTNF-RI with potential human MHCclass II binding activity are shown in Table 13.

Table 13. Potential T-cell epitopes of human 30KDa sTNF-RI.

TABLE 13 Potential T-cell epitopes of human 30KDa sTNF-RI DSVCPQGKYIHPQ,KYIHPQNNSICCT, NSICCTKCHKGTY, (SEQ ID NO: 309) (SEQ ID NO: 310) (SEQ IDNO: 311) TYLYNDCPGPGQD, YLYNDCPGPGQDT, NHLRHCLSCSKCR, (SEQ ID NO: 312)(SEQ ID NO: 313) (SEQ ID NO: 314) HCLSCSKCRKEMG, KEMGQVEISSCTV,GQVEISSCTVDRD, (SEQ ID NO: 315) (SEQ ID NO: 316) (SEQ ID NO: 317)VEISSCTVDRDTV, CTVDRDTVCGCRK, DTVCGCRKNQYRH, (SEQ ID NO: 318) (SEQ IDNO: 319) (SEQ ID NO: 320) NQYRHYWSENLFQ, RHYWSENLFQCFN, HYWSENLFQCFNC,(SEQ ID NO: 321) (SEQ ID NO: 322) (SEQ ID NO: 323) ENLFQCFNCSLCL,NLFQCFNCSLCLN, QCFNCSLCLNGTV, (SEQ ID NO: 324) (SEQ ID NO: 325) (SEQ IDNO: 326) CSLCLNGTVHLSC, LCLNGTVHLSCQE, GTVHLSCQEKQNT, (SEQ ID NO: 327)(SEQ ID NO: 328) (SEQ ID NO: 329) VHLSCQEKQNTVC, EKQNTVCTCHAGF,NTVCTCHAGFFLR, (SEQ ID NO: 330) (SEQ ID NO: 331) (SEQ ID NO: 332)GFFLRENECVSCS, FFLRENECVSCSN, ECVSCSNCKKSLE, (SEQ ID NO: 333) (SEQ IDNO: 334) (SEQ ID NO: 335) KSLECTKLCLPQI, TKLCLPQIENVKG, LCLPQIENVKGTE,(SEQ ID NO: 336) (SEQ ID NO: 337) (SEQ ID NO: 338) PQIENVKGTEDSG,SGTTVLLPLVIFF (SEQ ID NO: 339) (SEQ ID NO: 340)

Peptide sequences in a human 40KDa sTNF inhibitor with potential humanMHC class II binding activity are shown in Table 14.

Table 14. Potential T-cell epitopes of human 40KDa sTNF inhibitor.

TABLE 14 Potential T-cell epitopes of human 40KDa sTNF inhibitorTPYAPEPGSTCRL, CRLREYYDQTAQM, REYYDQTAQMCCS, (SEQ ID NO: 341) (SEQ IDNO: 342) (SEQ ID NO: 343) EYYDQTAQMCCSK, AQMCCSKCSPGQH, KCSPGQHAKVFCT,(SEQ ID NO: 344) (SEQ ID NO: 345) (SEQ ID NO: 346) AKVFCTKTSDTVC,KVFCTKTSDTVCD, STYTQLWNWVPEC, (SEQ ID NO: 347) (SEQ ID NO: 348) (SEQ IDNO: 349) TQLWNWVPECLSC, QLWNWVPECLSCG, NWVPECLSCGSRC, (SEQ ID NO: 350)(SEQ ID NO: 351) (SEQ ID NO: 352) FCLSCGSRCSSDQ, SRCSSDQEVTQAC,QEVTQACTREQNR, (SEQ ID NO: 353) (SEQ ID NO: 354) (SEQ ID NO: 355)QMRICTCRPGWYC, NRICTCRPGWYCA, PGWYCALSKQEGC, (SEQ ID NO: 356) (SEQ IDNO: 357) (SEQ ID NO: 358) GWYCALSKQEGCR, CALSKQEGCRLCA, APLRKCRPGFGVA,(SEQ ID NO: 359) (SEQ ID NO: 360) (SEQ ID NO: 361) PGFGVARPGTETS,FGVARPGTETSDV, SDVVCKPCAPGTF, (SEQ ID NO: 362) (SEQ ID NO: 363) (SEQ IDNO: 364) GTFSNTTSSTDIC, TDICRPHQICNVV, HQICNVVAIPGNA, (SEQ ID NO: 365)(SEQ ID NO: 366) (SEQ ID NO: 367) ICNVVAIPGNASR, CNVVAIPGNASRD,NVVAIPGNASRDA, (SEQ ID NO: 368) (SEQ ID NO: 369) (SEQ ID NO: 370)VAIPGNASRDAVC, DAVCTSTTTPTRS, TRSMAPGAVHLPQ, (SEQ ID NO: 371) (SEQ IDNO: 372) (SEQ ID NO: 373) RSMAPGAVHLPQP, VHLPQPVSTRSQH, QPVSTRSQHTQPT,(SEQ ID NO: 374) (SEQ ID NO: 375) (SEQ ID NO: 376) PEPSTAPSTSFLL,SFLLPMGPSPPAE, FLLPMGPSPPAEG (SEQ ID NO: 377) (SEQ ID NO: 378) (SEQ IDNO: 379)

EXAMPLE 9

(Soluble TNF-R2):

Fc-sTNF-R2 is a fusion proteins in which the serum half-life is extendedcompared to sTNF-R2 itself. However, certain forms of Fc-TNF-R2, such aswhen the Fc is derived from human IgG1 or human IgG3, have the potentialto show enhanced immunogenicity under certain circumstances, such asadministration by subcutaneous injection.

Soluble tumor necrosis factor receptor 2 (sTNF-R2) is a derivative ofthe human tumor necrosis factor receptor 2 described previously [Smith,C. A. et al (1990) Science 248: 1019–1023; Kohno, T. et al (1990) Proc.Nat. Acad. Sci. U.S.A. 87: 8331–8335; Beltinger, C. P. et al (1996)Genomics 35:94–100] comprising the extracellular domain of the intactreceptor. The soluble forms are able to bind tumour necrosis factor withhigh affinity and inhibit the cytotoxic activity of the cytokine invitro. Recombinant preparations of sTNF-R2 are of significanttherapeutic value for the treatment of diseases where an excess level oftumour necrosis factor is causing a pathogenic effect. A particularrecombinant preparation termed ethanercept has gained clinical approvalfor the treatment of rheumatoid arthritis and this and other similaragents may be of value in the treatment of other indications such ascachexia, sepsis and autoimmune disorders. Ethanercept is a dimericfusion protein comprising the extracellular domain of the human TNFR2molecule in combination with the Fc domain of the human IgG1 molecule.The dimeric molecule comprises 934 amino acids [U.S. Pat. No. 5,395,760;U.S. Pat. No. 5,605,690; U.S. Pat. No. 5,945,397].

Peptide sequences in the TNF binding domain of the human TNFR2 proteinwith potential human MHC class II binding activity are shown in Table15.

Table 15. Potential T-cell epitopes of human TNFR2 protein.

TABLE 15 Potential T-cell epitopes of human TNFR1 protein TPYAPEPGSTCRL,CRLREYYDQTAQM, REYYDQTAQMCCS, (SEQ ID NO: 380) (SEQ ID NO: 381) (SEQ IDNO: 382) EYYDQTAQMCCSK, AQMCCSKCSPGQH, KCSPGQHAKVFCT, (SEQ ID NO: 383)(SEQ ID NO: 384) (SEQ ID NO: 385) AKVFCTKTSDTVC, KVFCTKTSDTVCD,STYTQLWNWVPEC, (SEQ ID NO: 386) (SEQ ID NO: 387) (SEQ ID NO: 388)TQLWNWVPECLSC, QLWNWVPECLSCG, NWVPECLSCGSRC, (SEQ ID NO: 389) (SEQ IDNO: 390) (SEQ ID NO: 391) ECLSCGSRCSSDQ, SRCSSDQEVTQAC, QEVTQACTREQNR,(SEQ ID NO: 392) (SEQ ID NO: 393) (SEQ ID NO: 394) QNRICTCRPGWYC,NRICTCRPGWYCA, PGWYCALSKQEGC, (SEQ ID NO: 395) (SEQ ID NO: 396) (SEQ IDNO: 397) GWYCALSKQEGCR, CALSKQEGCRLCA, APLRKCRPGFGVA, (SEQ ID NO: 398)(SEQ ID NO: 399) (SEQ ID NO: 400) PGFGVARPGTETS, FGVARPGTETSDV,SDVVCKPCAPGTF, (SEQ ID NO: 401) (SEQ ID NO: 402) (SEQ ID NO: 403)GTFSNTTSSTDIC, TDICRPHQICNVV, HQICNVVAIPGNA, (SEQ ID NO: 404) (SEQ IDNO: 405) (SEQ ID NO: 406) ICNVVAIPGNASR, CNVVAIPCNASRD, NVVAIPGNASRDA,(SEQ ID NO: 407) (SEQ ID NO: 408) (SEQ ID NO: 409) VAIPGNASRDAVC,DAVCTSTTTPTRS, TRSMAPGAVHLPQ, (SEQ ID NO: 410) (SEQ ID NO: 411) (SEQ IDNO: 412) RSMAPGAVHLPQP, VHLPQPVSTRSQH, QPVSTRSQHTQPT, (SEQ ID NO: 413)(SEQ ID NO: 414) (SEQ ID NO: 415) PEPSTAPSTSFLL, SFLLPMGPSPPAE,FLLPMGPSPPAEG (SEQ ID NO: 416) (SEQ ID NO: 417) (SEQ ID NO: 418)

EXAMPLE 10

Non-Natural Forms of Beta-Glucocerebrosidase (β-GCR)

Fc-β-GCR is a fusion proteins in which the serum half-life is extendedcompared to the β-GCR itself. However, certain forms of Fc-β-GCR, suchas when the Fc is derived from human IgG1 or human IgG3, have thepotential to show enhanced immunogenicity under certain circumstances,such as administration by subcutaneous injection. The present inventionprovides for modified forms of human GCR, preferably Fc-β-GCR, with oneor more T-cell epitopes removed.

Beta-Glucocerebrosidase (b-D-glucosyl-N-acylsphingosine glucohydrolase,E. C. 3.2.1.45) is a monomeric glycoprotein of 497 amino acid residues.The enzyme catalyses the hydrolysis of the glycolipid glucocerebrosideto glucose and ceramide. Deficiency in GCR activity results in alysosomal storage disease referred to as Gaucher disease. The disease ischaracterised by the accumulation of glucocerebroside engorged tissuemacrophages that accumulate in the liver, spleen, bone marrow and otherorgans. The disease has varying degrees of severity from type 1 diseasewith haematologic problems but no neuronal involvement, to type 2disease manifesting early after birth with extensive neuronalinvolvement and is universally progressive and fatal within 2 years ofage. Type 3 disease is also recognised in some classifications and alsoshows neurologic involvement. Previously the only useful therapy forGaucher disease has been administration of GCR derived from humanplacenta (known as alglucerase) but more recently pharmaceuticalpreparations of recombinant GCR (“Ceredase” and “Cerezyme”) have shownefficacy in the treatment of type I disease [Niederau, C. et al (1998)Eur. J. Med. Res. 3: 25–30].

According to the invention, the particular commercial forms ofglucocerebrosidase are examined at predicted to be particularlyimmunogenic because these forms are engineered to have a high mannoseoligosaccharide. Upon administration of such a protein, such as Ceredaseor Cerezyme, the non-natural protein is preferentially bound by mannosereceptors on antigen-presenting cells such as macrophages or dendriticcells. The non-natural protein is then taken up, a portion is degradedinto peptides, and the peptides presented through MHC Class II to theT-cell receptor. By mutating the glucocerebrosidase sequence such thatderived peptides cannot bind to MHC Class II, immunogenicity is reduced.

Others have provided GCR molecules including modified GCR [U.S. Pat. No.5,236,838] but this teaching does not recognize the importance of T-cellepitopes to the immunogenic properties of the protein.

Peptide sequences in human b-GCR with potential human MHC class IIbinding activity are shown in Table 16.

Table 16. Potential T-cell epitopes of human human b-GCR.

TABLE 16 Potential T-cell epitopes of human hyman b-GCR PCIPKSFGYSSVV,KSFGYSSVVCVCN, FGYSSVVCVCNAT, SSVVCVCNATYCD, SVVCVCNATYCDS, (SEQ ID NO:419) (SEQ ID NO: 420) (SEQ ID NO: 421) (SEQ ID NO: 422) (SEQ ID NO: 423)VCVCNATYCDSFD, ATYCDSFDPPTFP, DSFDPPTFPALGT, PTFPALGTFSRYE,PALGTFSRYESTR, (SEQ ID NO: 424) (SEQ ID NO: 425) (SEQ ID NO: 426) (SEQID NO: 427) (SEQ ID NO: 428) GTFSRYESTRSGR, SRYESTRSGRRME,GRRMELSMGPIQA, RRMELSMGPIQAN, RMELSMGPIQANH, (SEQ ID NO: 429) (SEQ IDNO: 430) (SEQ ID NO: 431) (SEQ ID NO: 432) (SEQ ID NO: 433)MELSMGPIQANHT, LSMGPIQANHTGT, MGPIQANHTGTGL, GPIQANHTGTGLL,TGLLLTLQPEQKF, (SEQ ID NO: 434) (SEQ ID NO: 435) (SEQ ID NO: 436) (SEQID NO: 437) (SEQ ID NO: 438) GLLLTLQPEQKFQ, LLLTLQPEQKFQK,LTLQPEQKFQKVK, TLQPEQKFQKVKG, PEQKFQKVKGFGG, (SEQ ID NO: 439) (SEQ IDNO: 440) (SEQ ID NO: 441) (SEQ ID NO: 442) (SEQ ID NO: 443)QKFQKVKGFGGAM, QKVKGFGGAMTDA, KGFGGAMTDAAAL, GFGGAMTDAAALN,GAMTDAAALNILA, (SEQ ID NO: 444) (SEQ ID NO: 445) (SEQ ID NO: 446) (SEQID NO: 447) (SEQ ID NO: 448) AMTDAAALNILAL, MTDAAALNILALS,AALNILALSPPAQ, ALNILALSPPAQN, LNILALSPPAQNL, (SEQ ID NO: 449) (SEQ IDNO: 450) (SEQ ID NO: 451) (SEQ ID NO: 452) (SEQ ID NO: 453)NILALSPPAQNLL, LALSPPAQNLLLK, ALSPPAQNLLLKS, FAQNLLLKSYFSE,AQNLLLKSYFSEE, (SEQ ID NO: 454) (SEQ ID NO: 455) (SEQ ID NO: 456) (SEQID NO: 457) (SEQ ID NO: 458) QNLLLKSYFSEEG, NLLLKSYFSEEGI,LLLKSYFSEEGIG, KSYFSEEGIGYNI, SYFSEEGIGYNII, (SEQ ID NO: 459) (SEQ IDNO: 460) (SEQ ID NO: 461) (SEQ ID NO: 462) (SEQ ID NO: 463)FSEEGIGYNIIRV, EGIGYNIIRVPMA, GIGYNIIRVPMAS, IGYNIIRVPMASC,YNIIRVPMASCDF. (SEQ ID NO: 464) (SEQ ID NO: 465) (SEQ ID NO: 466) (SEQID NO: 467) (SEQ ID NO: 468) NIIRVPMASCDFS, IIRVPMASCDFSI,IRVPMASCDFSIR, VPMASCDFSIRTY, PMASCDFSIRTYT, (SEQ ID NO: 469) (SEQ IDNO: 470) (SEQ ID NO: 471) (SEQ ID NO: 472) (SEQ ID NO: 473)SCDFSIRTYTYAD, CDFSIRTYTYADT, FSIRTYTYADTPD, RTYTYADTPDDFQ,TYTYADTPDDFQL, (SEQ ID NO: 474) (SEQ ID NO: 475) (SEQ ID NO: 476) (SEQID NO: 477) (SEQ ID NO: 478) YTYADTPDDFQLH, ADTPDDFQLHNFS,PDDFQLHNFSLPE, DDFQLHNFSLPEE, FQLHNFSLPEEDT, (SEQ ID NO: 479) (SEQ IDNO: 480) (SEQ ID NO: 481) (SEQ ID NO: 482) (SEQ ID NO: 483)HNFSLPEEDTKLK, FSLPEEDTKLKIP, SLPEEDTKLKIPL, EEDTKLKIPLIHR,TKLKIPLIHRALQ, (SEQ ID NO: 484) (SEQ ID NO: 485) (SEQ ID NO: 486) (SEQID NO: 487) (SEQ ID NO: 488) KLKIPLIHRALQL, LKIPLIHRALQLA,IPLIHRALQLAQR, PLIHRALQLAQRP, HRALQLAQRPVSL, (SEQ ID NO: 489) (SEQ IDNO: 490) (SEQ ID NO: 491) (SEQ ID NO: 492) (SEQ ID NO: 493)RALQLAQRPVSLL, ALQLAQRPVSLLA, LQLAQRPVSLLAS, RPVSLLASPWTSP,PVSLLASPWTSPT, (SEQ ID NO: 494) (SEQ ID NO: 495) (SEQ ID NO: 496) (SEQID NO: 497) (SEQ ID NO: 498) VSLLASPWTSPTW, SLLASPWTSPTWL,SPWTSPTWLKTNG, TSPTWLKTNGAVN, PTWLKTNGAVNGK, (SEQ ID NO: 499) (SEQ IDNO: 500) (SEQ ID NO: 501) (SEQ ID NO: 502) (SEQ ID NO: 503)TWLKTNGAVNGKG, GAVNGKGSLKGQP, GSLKGQPGDIYHQ, GDIYHQTWARYFV,DIYHQTWARYFVK, (SEQ ID NO: 504) (SEQ ID NO: 505) (SEQ ID NO: 506) (SEQID NO: 507) (SEQ ID NO: 508) QTWARYFVKFLDA, WARYFVKFLDAYA,ARYFVKFLDAYAE, RYFVKFLDAYAEH, YFVKFLDAYAEHK, (SEQ ID NO: 509) (SEQ IDNO: 510) (SEQ ID NO: 511) (SEQ ID NO: 512) (SEQ ID NO: 513)FVKFLDAYAEHKL, VKFLDAYAEHKLQ, KFLDAYAEHKLQF, DAYAEHKLQFWAV,YAEHKLQFWAVTA, (SEQ ID NO: 514) (SEQ ID NO: 515) (SEQ ID NO: 516) (SEQID NO: 517) (SEQ ID NO: 518) HKLQFWAVTAENE, LQFWAVTAENEPS,QFWAVTAENEPSA, FWAVTAENEPSAG, WAVTAENEPSAGL, (SEQ ID NO: 519) (SEQ IDNO: 520) (SEQ ID NO: 521) (SEQ ID NO: 522) (SEQ ID NO: 523)VTAENEPSAGLLS, PSAGLLSGYPFQC, AGLLSGYPFQCLG, GLLSGYPFQCLGF,SGYPFQCLGFTPE, (SEQ ID NO: 524) (SEQ ID NO: 525) (SEQ ID NO: 526) (SEQID NO: 527) (SEQ ID NO: 528) YPFQCLGFTPEHQ, QCLGFTPEHQRDF,LGFTPEHQRDFIA, FTPEHQRDFIARD, RDFIARDLGPTLA, (SEQ ID NO: 529) (SEQ IDNO: 530) (SEQ ID NO: 531) (SEQ ID NO: 532) (SEQ ID NO: 533)DFIARDLGPTLAN, RDLGPTLANSTHH, LGPTLANSTHHNV, PTLANSTHHNVRL,HNVRLLMLDDQRL, (SEQ ID NO: 534) (SEQ ID NO: 535) (SEQ ID NO: 536) (SEQID NO: 537) (SEQ ID NO: 538) VRLLMLDDQRLLL, RLLMLDDQRLLLP,LLMLDDQRLLLPH, LMLDDQRLLLPHW, DDQRLLLPHWAKV, (SEQ ID NO: 539) (SEQ IDNO: 540) (SEQ ID NO: 541) (SEQ ID NO: 542) (SEQ ID NO: 543)DQRLLLPHWAKVV, QRLLLPHWAKVVL, RLLLPHWAKVVLT, LLLPHWAKVVLTD,PHWAKVVLTDPEA, (SEQ ID NO: 544) (SEQ ID NO: 545) (SEQ ID NO: 546) (SEQID NO: 547) (SEQ ID NO: 548) WAKVVLTDPEAAK, AKVVLTDPEAAKY,KVVLTDPEAAKYV, VVLTDPEAAKYVH, EAAKYVIGIAVHW, (SEQ ID NO: 549) (SEQ IDNO: 550) (SEQ ID NO: 551) (SEQ ID NO: 552) (SEQ ID NO: 553)AKYVHGIAVHWYL, KYVHGIAVHWYLD, YVHGIAVHWYLDF, HGIAVHWYLDFLA,IAVHWYLDFLAPA, (SEQ ID NO: 554) (SEQ ID NO: 555) (SEQ ID NO: 556) (SEQID NO: 557) (SEQ ID NO: 558) VNWYLDFLAPAKA, HWYLDFLAPAKAT,WYLDFLAPAKATL, LDFLAPAKATLGE, DFLAPAKATLGET, (SEQ ID NO: 559) (SEQ IDNO: 560) (SEQ ID NO: 561) (SEQ ID NO: 562) (SEQ ID NO: 563)AKATLGETHRLFP, ATLGETHRLFPNT, GETHRLFPNTMLF, ETHRLFPNTMLFA,THRLFPNTMLFAS, (SEQ ID NO: 564) (SEQ ID NO: 565) (SEQ ID NO: 566) (SEQID NO: 567) (SEQ ID NO: 568) HRLFPNTMLFASE, RLFPNTMLFASEA,FPNTMLFASEACV, NTMLFASEACVGS, TMLFASEACVGSK, (SEQ ID NO: 569) (SEQ IDNO: 570) (SEQ ID NO: 571) (SEQ ID NO: 572) (SEQ ID NO: 573)MLFASEACVGSKF, ACVGSKFWEQSVR, GSKFWEQSVRLGS, SKFWEQSVRLGSW,KFWEQSVRLGSWD, (SEQ ID NO: 574) (SEQ ID NO: 575) (SEQ ID NO: 576) (SEQID NO: 577) (SEQ ID NO: 578) QSVRLGSWDRGMQ, VRLGSWDRGMQYS,RLGSWDRGMQYSH, GSWDRGMQYSHSI, WDRGMQYSHSIIT, (SEQ ID NO: 579) (SEQ IDNO: 580) (SEQ ID NO: 581) (SEQ ID NO: 582) (SEQ ID NO: 583)RGMQYSHSIITNL, MQYSHSIITNLLY, QYSHSIITNLLYH, YSHSIITNLLYHV,HSIITNLLYHVVG, (SEQ ID NO: 584) (SEQ ID NO: 585) (SEQ ID NO: 586) (SEQID NO: 587) (SEQ ID NO: 588) SIITNLLYHVVGW, TNLLYHVVGWTDW,NLLYHVVGWTDWN, LLYHVVGWTDWNL, YHVVGWTDWNLAL, (SEQ ID NO: 589) (SEQ IDNO: 590) (SEQ ID NO: 591) (SEQ ID NO: 592) (SEQ ID NO: 593)HVVGWTDWNLALM, VVGWTDWNLALNP, VGWTDWNLALNPE, TDWNLALNPEGGP,WNLALNPEGGPNW, (SEQ ID NO: 594) (SEQ ID NO: 595) (SEQ ID NO: 596) (SEQID NO: 597) (SEQ ID NO: 598) LALNPEGGPNWVR, PNWVRNFVDSPII,NWVRNFVDSPIIV, RNFVDSPIIVDIT, NFVDSPIIVDITK, (SEQ ID NO: 599) (SEQ IDNO: 600) (SEQ ID NO: 601) (SEQ ID NO: 602) (SEQ ID NO: 603)SPIIVDITKDTFY, PIIVDITKDTFYK, IIVDITKDTFYKQ, VDITKDTFYKQPM,DTFYKQPMFYHLG, (SEQ ID NO: 604) (SEQ ID NO: 605) (SEQ ID NO: 606) (SEQID NO: 607) (SEQ ID NO: 608) TFYKQPMFYHLGH, QPMFYHLCHFSKF,PMFYHLGHFSKPI, MFYHLGHFSKFIP, YHLGHFSKFIPEG, (SEQ ID NO: 609) (SEQ IDNO: 610) (SEQ ID NO: 611) (SEQ ID NO: 612) (SEQ ID NO: 613)GHFSKFIPEGSQR, SKFIPEGSQRVGL, KFIPEGSQRVGLV, IPEGSQRVGLVAS,QRVGLVASQKNDL, (SEQ ID NO: 614) (SEQ ID NO: 615) (SEQ ID NO: 616) (SEQID NO: 617) (SEQ ID NO: 618) VGLVASQKNDLDA, GLVASQKNDLDAV,SQKNDLDAVALMH, NDLDAVALMHPDG, DAVALMHPDGSAV, (SEQ ID NO: 619) (SEQ IDNO: 620) (SEQ ID NO: 621) (SEQ ID NO: 622) (SEQ ID NO: 623)VALMHPDGSAVVV, ALMHPDGSAVVVV, SAVVVVLNRSSKD, AVVVVLNRSSKDV,VVVVLNRSSKDVP, (SEQ ID NO: 624) (SEQ ID NO: 625) (SEQ ID NO: 626) (SEQID NO: 627) (SEQ ID NO: 628) VVVLNRSSKDVPL, VVLNRSSKDVPLT,KDVPLTIKDPAVG, VPLTIKDPAVGFL, PLTIKDPAVGFLE, (SEQ ID NO: 629) (SEQ IDNO: 630) (SEQ ID NO: 631) (SEQ ID NO: 632) (SEQ ID NO: 633)LTIKDPAVGFLET, PAVGFLETISPGY, VGFLETISPGYSI, GFLETISPGYSIH,FLETISPGYSIHT, (SEQ ID NO: 634) (SEQ ID NO: 635) (SEQ ID NO: 636) (SEQID NO: 637) (SEQ ID NO: 639) ETISPGYSIHTYL, PGYSIHTYLWHRQ, PGYSIHTYLWRRQ(SEQ ID NO: 639) (SEQ ID NO: 640) (SEQ ID NO: 641)

EXAMPLE 11

De-immunized Forms of Fc-IL2

Non-deimmunized Fc-IL2 was described e.g. in WO 96/08570. Specificde-immunized Fc-IL2 forms: Fcγ1-IL2, Fcγ2-IL2, both forms, preferablywith linker peptide and optionally modified Fc domain having reducedaffinity to Fc-receptors.

EXAMPLE 12

De-immunized Forms Fc-IL12

Non-deimmunized Fc-IL12 was described e.g. in WO 99/29732. Specificde-immunized Fc-IL12 forms: Fcγ1-IL12, Fcγ2-IL12, both forms, preferablywith linker peptide and optionally modified Fc domain having reducedaffinity to Fc-receptors.

EXAMPLE 13

De-immunized Forms of Fc-TNFα

Non-deimmunized Fc-TNFa was described e.g. in WO 99/43713. Specificde-immunized Fc-TNFa forms: Fcγ1-TNFa, Fcγ2-TNFa, both forms, preferablywith linker peptide and optionally modified Fc domain having reducedaffinity to Fc-receptors.

EXAMPLE 14

De-immunized Forms of Fc-GM-CSF

Non-deimmunized Fc-GM-CSF was described e.g. in WO 99/43713 and WO01/07081. Specific de-immunized Fc-GM-CSF forms: Fcγ1-GM-CSF,Fcγ2-GM-CSF, both forms, preferably with linker peptide and optionallymodified Fc domain having reduced affinity to Fc-receptors.

EXAMPLE 15

De-immunized Forms of Fc-Subtilisin

Specific de-immunized Fc-subtilisin forms: Fcγ1-subtilisin,Fcγ2-subtilisin, both forms, preferably with linker peptide andoptionally modified Fc domain having reduced affinity to Fc-receptors.

EXAMPLE 16

De-immunized forms of Fc-Insulin

Specific de-immunized Fc-insulin forms: Fcγ1-insulin, Fcγ2-insulin, bothforms, preferably with linker peptide and optionally modified Fc domainhaving reduced affinity to Fc-receptors.

EXAMPLE 17

De-immunized Forms of Fc-PSMA

Non-deimmunized Fc-PSMA was described e.g. in WO 96/08570 and WO01/0708. Specific de-immunized Fc-PSMA forms: Fcγ1-PSMA, Fcγ2-PSMA, bothforms, preferably with linker peptide and optionally modified Fc domainhaving reduced affinity to Fc-receptors.

EXAMPLE 18

De-immunized Fusion Proteins Comprising Anti-EGFR Antibodies Fused to aCytokine

Humanized and murine monoclonal antibody 425 (hMAb 425, U.S. Pat. No.5,558,864; EP 0531 472), murine and chimeric monoclonal antibody 225(cMAb 225, U.S. Pat. No. 4,943,533 and EP 0359 282) and murine andhumanized MAb 4D5 (hMab 4D5=Herceptin®) have been de-immunized accordingto the invention and fused to a de-immunized IL2 or a non-modified IL-2.Fusions of antibodies to cytokines represent a situation where the needto reduce immunogenicity is particularly great. Normally, therapeuticantibodies can induce anti-idiotype antibodies that neutralize theeffectiveness of a therapeutic antibody. This is particularly true whena therapeutic antibody is administered at low or medium levels, asopposed to very high levels where tolerance can be induced. For example,the therapeutic antibodies Herceptin and Rituxan are generally given inhigh doses of a few hundred milligrams. In contrast, antibody-cytokinefusions are generally given in a lower dose on the order of a fewmilligrams. Thus, the dose of an antibody-cytokine fusion is in therange that tends to promote formation of anti-idiotype antibodies. Thepresence of the linked cytokine tends to exaggerate the immunogenicityof the already immunogenic antibodies.

Antibody 425 is a non-human antibody which is directed to antigen EGF-Rand reacts with colon cancer cells. This antibody has been fused toIL-2, as described in Example 13. The presence of IL-2 or anothercytokine enhances the immunogenicity of the antibody, in particular theV regions.

In the following paragraphs the invention is described in more detailfor the monoclonal anti-EGFR antibody 425-IL2 construct which was shownto have a high therapeutic value. However, the invention is not limitedto this antibody and said construct and its several existing forms, butcan be extended to other anti-EGER antibodies and their fusionconstructs, preferably cytokine fusion immunoglobulins, above allchimeric antibody 225 (c225-Il-2), which has very similar properties. Inprincipal, non-human, chimeric or humanized versions of the anti-EGFRantibodies can be used to synthesize said IL-2 fusion molecules Unlessstated otherwise all amino acids in the variable heavy and light chainsare numbered as in Kabat et al, 1991 (Sequences of Proteins ofImmunological Interest, US Department of Health and Human Services).Potential T-cell epitopes are numbered with the linear number of thefirst amino acid of an epitope, counting from the first amino acid ofthe heavy and light chains.

1. Comparison with Mouse Subgroup Frameworks

The amino acid sequences of murine 425 VH (heavy chain) and VK (lightchain) were compared to consensus sequences for the Kabat murine heavyand light chain subgroups. 425 VH can be assigned to mouse heavy chainssubgroup IIB. The comparison with the consensus sequence for thissubgroup shows that the serine at position 94 in 425 VH is unusual. Themost common residue at this position is arginine. 425 VK can be assignedto mouse kappa chains subgroup VI. The comparison with the consensussequence for this subgroup shows that the residues at positions 45–47,60 and 100 in 425 VK are unusual for this subgroup. Amino acid residuenumbering is as per Kabat.

2. Comparison with Human Frameworks

The amino acid sequences of murine 425 VH (variable heavy chain) and VK(variable kappa light chain) were compared to the sequences of thedirectory of human germline VH (Tomlinson, I. M et al., (1992) J. Mol.Biol. 227: 776–798) and VK (Cox, J. P. L. et al., (1994) Eur. J.Immunol. 24:827–836) sequences and also to human germline J regionsequences (Routledge, E. G. et al., in, Protein Engineering of AntibodyMolecules for Prophylactic and Therapeutic Applications in Man, Clark,M. ed. Academic Titles, Nottingham, UK, pp 13–44, 1991). The murine 425sequence of the heavy and light chain can be taken, for example, from EP0531 472.

The reference human framework selected for 425 VH was VH1GRR with humanJH6. The sequence of VH1GRR in the directory ends at residue 88.Therefore there is no corresponding residue for the unusual serine atposition 94 of the murine sequence. This germline sequence has beenfound in a rearranged mature antibody gene with 4 amino acid changes.The reference human framework selected for 425 VK was L6/vg with humanJK2. This germline sequence has been found in a rearranged matureantibody heavy chain with no amino acid changes.

3. Design of “Veneered” Sequences

Following identification of the reference human framework sequences,certain non-identical amino acid residues within the 425 VH and VKframeworks were changed to the corresponding amino acid in the humanreference framework sequence. Residues which are considered to becritical for antibody structure and binding were excluded from thisprocess and not altered: The murine residues that were retained at thisstage are largely non-surface, buried residues, apart from residues atthe N-terminus for instance, which are close to the CDRs in the finalantibody (1–8, preferably 1–5 amino acid residues). This processproduces a sequence that is broadly similar to a “veneered” antibody asthe surface residues are mainly human and the buried residues are as inthe original murine sequence.

4. Peptide Threading Analysis

The murine and veneered 425 VH and VK sequences were analyzed using themethod according of the invention. The amino acid sequences are dividedinto all possible 13 mers. The 13 mer peptides are sequentiallypresented to the models of the binding groove of the HLA-DR allotypesand a binding score assigned to each peptide for each allele. Aconformational score is calculated for each pocket-bound side chain ofthe peptide. This score is based on steric overlap, potential hydrogenbonds between peptide and residues in the binding groove, electrostaticinteractions and favorable contacts between peptide and pocket residues.The conformation of each side chain is then altered and the scorerecalculated. Having determined the highest conformational score, thebinding score is then calculated based on the groove-bound hydrophobicresidues, the non-groove hydrophobic residues and the number of residuesthat fit into the binding groove. Peptides which are known binders tohuman MHC Class II achieve a high binding score with almost no falsenegatives. Thus peptides that achieve a significant binding score in thecurrent analysis are considered to be potential T-cell epitopes. Theresults of the peptide threading analysis are shown in Table 17 for 425VH and 425 VK. Potential T Cell epitopes are referred to by the linearnumber of the first residue of the 13 mer.

Table 17. Potential T-cell epitopes in murine and veneered 425sequences.

TABLE 17 Potential T-cell epitopes in murine and veneered 425 sequencesNumber of potential T-cell Number of first residue of 13 mer withSequence epitopes number of bonding alleles in brackets Murine 8 31 (7),35 (17), 43 (7), 46 (8), 58 (10), 425 VH 62 (12), 81 (11), 84 (16)Veneered 7 31 (7), 43 (7), 46 (8), 58 (10), 62 (11), 425 VH 81 (11), 84(16) Murine 9 1 (8), 2 (5), 17 (5), 27 (5), 43 (16), 72 (18), 425 VK 75(10), 92 (10), 93 (17) Veneered 4 27 (5), 43 (16), 92 (8), 93 (17) 425VK

5. Removal of Potential T Cell Epitopes

The numbering of amino acid residues for substitution is as per Kabat.Potential T Cell epitopes are referred to by the linear number of thefirst residue of the 13 mer.

The amino acid substitutions required to remove the potential T-cellepitopes from the veneered 425 heavy chain variable region were asfollows:

-   -   Substitution of proline for alanine at residue 41 (Kabat        number 41) removes the potential epitope at residue number 31.    -   Substitution of proline for leucine at residue 45 (Kabat        number 45) removes the to potential epitope at residue        number 43. A proline at position 45 is found in a human germline        VH sequence, DP52.        -   Substitution of alanine for isoleucine at residue 48 (Kabat            number 48) removes the potential epitope at residue number            46.    -   Substitution of valine for alanine at residue 68 (Kabat        number 67) removes the potential epitope at residue number 58.    -   Substitution of isoleucine for leucine at residue 70 (Kabat        number 69) removes the potential epitope at residue number 62.    -   Substitution of threonine for serine at residue 91 (Kabat        number 87) removes the potential epitopes at residue numbers 81        and 84.

The amino acid substitutions required to remove the potential T-cellepitopes from the veneered 425 light chain variable region were asfollows:

-   -   Substitution of histidine for tyrosine at residue 35 (Kabat        number 36) removes the potential epitope at residue number 27        -   Substitution of alanine for threonine at residue 50 (Kabat            number 51) removes the potential epitope at residue            number 43. This residue is within CDR2. Alanine is commonly            found at this position in both human and murine antibodies.            An alternative substitution to eliminate this epitope is            alanine for leucine at position 45 (Kabat number 46). There            is no conservative substitution that will eliminate the            potential epitope. Alanine is found at this position in some            antibodies.    -   Substitution of proline for isoleucine at residue 94 (Kabat        number 95) removes the potential epitope at residue number 92.        Kabat residue 95 is within CDRL3. Proline is common at this        position in mouse antibody sequences and there is no change        outwith the CDR that eliminates the potential epitope.    -   Substitution of valine for leucine at residue 103 (Kabat        number 104) removes the potential epitope at residue number 93.        6. Design of De-immunized Sequences

De-immunized heavy and light chain variable region sequences weredesigned with reference to the changes required to remove potentialT-cell epitopes and consideration of framework residues that might becritical for antibody structure and binding. In addition to theDe-immunized sequences based on the veneered sequence, an additionalsequence was designed for each of VH and VK based on the murinesequence, termed the Mouse Peptide Threaded (MoPT) version. For thisversion, changes were made directly to the murine sequence in order toeliminate T-cell epitopes, but only changes out with the CDRs that arenot considered to be detrimental to binding are made. No attempt toremove surface (B-cell) epitopes has been made in this version of thede-immunized sequence.

The primary de-immunized VII includes substitutions 1 to 6 in Section 5above and includes no potential T-cell epitopes. A further 4de-immunized VH sequences were designed in order to test the effect ofthe various substitutions required on antibody binding. The cumulativealterations made to the primary de-immunized sequence (425 VH1GRR-VH-v1)and the potential T-cell epitopes remaining are detailed in Table 18.The mouse threaded version is included for comparison.

Table 18. Amino acid changes and potential epitopes in de-immunized 425VH.

TABLE 18 Amino acid changes and potential epitopes in de-immunized 425VH Cumulative Variant Residue Changes Potential T Cell Epitopes 425VH1GRR-VH-v1 None None 425 VH1GRR-VH-v2 48A → I 46 (8) 425 VH1GRR-VH-v345P → L 43 (7), 46 (8) 425 VH1GRR-VH-v4 67V → A, 43 (7), 46 (8), 69I → L58 (10), 62 (11) 425 VH1GRR-VH-v5 41P → A 31 (7), 43 (7), 46 (8), 58(10), 62 (11) 425 VH-MoPT NA 43 (7), 46 (8)

The primary de-immunized VK includes substitutions 1 to 4 in Section 5above and includes no potential T-cell epitopes. A further 4de-immunized VK sequences were designed in order to test the effect ofthe various substitutions required on antibody binding. Version 2 is analternative to Version 1 in which an alternative substitution has beenused to remove the same potential T-cell epitope. The cumulativealterations made to the primary de-immunized sequence (425 L6-vg-VK-v1)and the potential T-cell epitopes remaining are detailed in Table 19.The mouse threaded version is included for comparison. Original andveneered sequences of VH and VK of murine MAb 425 are shown in Table 20.De-immunized sequences of the variable and heavy chains of MAb 425 areshown in Table 21.

Table 19. Amino acid changes and potential epitopes in de-immunized 425VK.

TABLE 19 Amino acid changes and potential epitopes in de-immunized 425VK Cumulative Variant Residue Changes Potential T-cell Epitopes 425L6-vg-VK-v1 None None 425 L6-vg-VK-v1 51 A → T, 46L → A None 425L6-vg-VK-v1 46 A → L 43 (16) 425 L6-vg-VK-v1 95 P → I 43 (16), 92 (8)425 L6-vg-VK-v1 36 H → Y 27 (5), 43 (16), 92 (8) 425 VK-MoPT NA 27 (5),43 (16), 92 (8)

Table 20. Original and veneered sequences of VH and VK of murine MAb425.

TABLE 20 original and “veneered” sequences of VH and VK of murine MAb425 425 VH mouse (SEQ ID NO: 642):QVQLQQPGAELVKPGASVKLSCKASGYTFTSHWMHWVKQRAGQGLEWIGEFNPSNGRTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCASRDYDYDGRYFDYWGQGTTLTVSS 425 VK mouse (SEQID NO: 643):QIVLTQSPAIMSASPGEKVTMTCSASSSVTYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSHIFTFGSGTKLEIK 425 VH veneered: (SEQ ID NO: 644):QVQLVQSGAELVKPGASVKLSCKASGYTFTSHWMHWVKQAAGQGLEWIGEFNPSNGRTNYNEKFKSRATLTVDKSTSTAYMQLSSLTSEDSAVYYCASRDYDYDGRYFDYWGQGTTLTVSS 425 VKveneered:(SEQ ID NO: 645):QIVLTQSPATLSASPGERATMSCSASSSVTYMYWYQQKPGQSPRLLIYDTSNLASGVPARFSGSGSGTSYTLTISSLEAEDAATYYCQQWSSHIFTFGQGTKLEIK

Table 21. De-immunized sequences of variable heavy and light chain ofMAb 425.

TABLE 21 De-immunized sequences of variable heavy and light chain of MAb425 425 de-immunized VH1 (SEQ ID NO: 646):QVQLVQSGAELVKPGASVKSCKASGYTFTSHWMHWVKQAPGQGPEWAGEFNPSNGRTNYNEKFKSRVTITVDKSTSTAYMQLSSLTSEDTAVYYCASRDYDYDGRYFDYWGQGTTLTVSS 425 de-immunizedVK1 (SEQ ID NO: 647):QIVLTQSPATLSASPGERATMSCSASSSVTYMYWHQQKPGQSPRLLIYDASNLASGVPARFSGSGSGTSYTLTISSLEAEDAATYYCQQWSSHPFTFGQGTKVEIK 425 de-immunized VH2 (SEQ ID NO:648):QIVLTQSPATLSASPGERATMSCSASSSVTYMYWHQQKPGQSPRALIYDTSNLASGVPARFSGSGSGTSITVDKSTSTAYMQL8SSLTSEDTAVYYCASRDYDYDGRYFDYWGQGTTLTVSS 425 de-immunizedVK2 (SEQ ID NO: 649):QIVLTQSPATLSASPGERATMSCSASSSVTYMYWHQQKPGQSPRALIYDTSNLASGVPARFSGSGSGTSYTLTISSLEAEDAATYYCQQWSSHPFTFGQGTKVEIK 425 de-immunized VH3 (SEQ ID NO:650):QVQLVQSGAELVKPGASVKLSCKASGYTFTSHWMHWVKQAPGQGLEWIGEFNPSNGRTNYNEKFKSRVTITVDKSTSTAYMQLSSLTSEDTAVYYCASRDYDYDGRYFDYWGQGTTLTVSS 425 de-immunizedVK3 (SEQ ID NO: 651):QIVLTQSPATLSASPGERATMSCSASSSVTYMYWHQQKPGQSPRLLIYDTSNLASGVPARFSGSGSGTSYTLTISSLEAEDAATYYCQQWSSHPFTFGQGTKVEIK 425 de-immunized VH4 (SEQ ID NO:652):QVQLVQSGAELVKPGASVKLSCKASGYTFTSHWMHWVKQAPGQGLEWIGEFNPSNGRTNYNEKFKSRATLTVDKSTSTAYMQLSSLTSEDTAVYYCASRDYDYDGRYFDYWGQGTTLTVSS 425 de-immunizedVK4 (SEQ ID NO: 653):QIVLTQSPATLSASPGERATMSCSASSSVTYNYWHQQKPGQSPRLLIYDTSNLASGVPARFSGSGSGTSYTLTISSLEAEDAATYYCQQWSSHIFTFGQGTKVEIK 425 de-immunized VH5 (SEQ ID NO:654):QVQLVQSGAELVKPGASVKLSCKASGYTFTSHWMHWVKQAAGQGLEWIGEENPSNGRTNYNEKFKSRATLTVDKSTSTAYMQLSSLTSEDTAVYYCASRDYDYDGRYFDYWGQGTTLTVSS 425 de-immunizedVK5 (SEQ ID NO: 655):QIVLTQSPATLSASPGERATMSCSASSSVTYMYWYQQKPGQSPRLLIYDTSNLASGVPARFSGSGSGTSYTLTISSLEAEDAATYYCQQWSSHIFTFGQGTKVEIK 425 VH mouse, peptide threaded (MoPT) (SEQ ID NO: 656):QVQLQQPGAELVKPGASVKLSCKASGYTFTSHWMHWVKQAPGQGLEWIGEFNPSNGRTNYMEKFKSRVTITVDKSSSTAYMQLSSLTSEDTAVYYCASRDYDYDGRYFDYWGQGTTLTVSS 425 VK mouse,peptide threaded (Mo PT) (SEQ ID NO: 657):QIVLTQSPATLSASPGEKATMTCSASSSVTYMYWYQQKPGSSPRLLIIYDTSNLASGVPVRFSGSGSGTSYSLTISRLEAEDAATYYCQQWSSHIFTFGQGTKVEIK

As already mentioned, the modified anti-EGFR antibody—cytokineconstructs according to the invention, preferably MAb 425-Il2, can beused in pharmaceutical compositions and pharmaceutical kits preferablyfor the treatment of cancer. “Cancer” and “tumor” refer to or describethe physiological condition in mammals that is typically characterizedby unregulated cell growth. By means of the pharmaceutical compositionsaccording of the present invention tumors can be treated such as tumorsof the breast, heart, lung, small intestine, colon, spleen, kidney,bladder, head and neck, ovary, prostate, brain, pancreas, skin, bone,bone marrow, blood, thymus, uterus, testicles, cervix, and liver.

In analogy to antibody 425 similar fusion constructs can be obtainedusing monoclonal antibody 225 in murine, chimeric or humanized forms.

EXAMPLE 19

De-immunized Forms of 14.18 Antibody-IL2 and KS-1/4-IL2

The cytokine interleukin 2 (IL-2) has been fused to specific monoclonalantibodies KS-1/4 and ch14.18 directed to the tumor associated antigensepithelial cell adhesion molecule (Ep-CAM, KSA, KS 1/4 antigen) and thedisialoganglioside GD, respectively, to form the fusion proteinsch14.18-IL-2 and KS1/4-IL-2, respectively, (U.S. Pat. No. 5,650,150, EP0 338 767). Theses antibodies have been de-immunized according to theinvention and fused to a immunogenicly modified IL2 or a non-modifiedIL-2.

Anti-EpCAM Antibody KS 1/4

The monoclonal antibody KS 1/4 is a murine antibody that specificallybinds to the 40,000 dalton cell surface antigen EpCAM (epithelial celladhesion molecule) found in high density on adenocarcinoma cells andalso found at much lower levels on certain normal epithelial cells. Thisantibody has been shown to be effective for the detection of disease.

A variety of fusions of KS-1/4 to single and combined cytokines such asIL-2 and IL-12, have been described (WO98/25978, WO01/58957A, and WO01/10912). These fusion proteins are effective in animal models ofcancer. However, due to the presence of cytokines, these fusion proteinsare particularly immunogenic. There is a need for altered KS antibodymolecules with a reduced propensity to elicit an immune response onadministration to the human host. Modified sequences in Mab KS 1/4providing a modified KS antibody according to the invention are shownbelow. A mutated form of the KS-1/4 in which the T-cell epitopes in theV regions were completely removed by mutation, as defined by thecriteria given above in the section on computer algorithms, wasefficiently expressed in mammalian cells and bound to the EpCAM antigenwith only about an 8-fold reduction of affinity. This molecule wastermed VHv1/VKv1. A second antibody, VHv2/VKv1, had only about a 3-foldreduction in affinity and differed from VHv1/VKv1 by a single amino acidsubstitution. These two antibodies have been expressed in mammaliancells as KS-IL2 fusion proteins. The KS(VHv1/VKv1)-IL2 and KS(VHv2/VKv1)are the most preferred embodiments of the invention with respect totreatment of a broad spectrum of human cancers by immune therapy.

1 Comparison with Mouse Subgroup Frameworks

The amino acid sequences of murine KS VH and VK were compared toconsensus sequences for the Kabat murine heavy and light chain subgroups(Kabat et al., 1991). Murine KS VH cannot be assigned to any oneSubgroup, but is closest to Subgroup II(A) and V(A). Unusual residuesare found at position 2 which is normally valine, 46 which is normallyglutamic acid, and 68 which is normally threonine. Residue 69 is morecommonly leucine or iso-leucine. At 82b, serine is most often found.Murine KS VK can be assigned to Subgroup VI (FIG. 2). Unusual residuesare found at 46 and 47 which are commonly both leucine. Residue 58 isunusual with either leucine or valine normally found at this position.

2 Comparison with Human Frameworks

The amino acid sequences of murine KS VH and VK were compared to thesequences of the directory of human germline VH (Tomlinson et al., 1992)and VK (COX et al. 1994) sequences and also to human germline J regionsequences (Routledge et al., 1993). The reference human frameworkselected for KS VH was DP10 with human JH6. This germline sequence hasbeen found in a rearranged mature antibody gene with no amino acidchanges. The reference human framework selected for KS VK was B1. Forframework-2 the sequence of the mature human antibody IMEV was used (inKabat et al 1991). This sequence is identical to the murine sequenceimmediately adjacent to CDR2. The J region sequence was human JK4. Thisgermline sequence has not been found as rearranged mature antibody lightchain.

3 Design of Veneered Sequences

Following identification of the reference human framework sequences,certain non-identical amino acid residues within the 425 VH and VKframeworks were changed to the corresponding amino acid in the humanreference sequence. Residues which are considered to be critical forantibody structure and bindin2 were excluded from this process and notaltered. The murine residues that were retained at this stage arelargely non-surface, buried residues, apart from residues at theN-terminus for instance, which are close to the CDRs in the finalantibody. This process produces a sequence that is broadly similar to a“veneered” antibody as the surface residues are mainly human and theburied residues are as in the original murine sequence.

4 Peptide Threading Analysis

The murine and veneered KS VH and VK sequences were analyzed using themethod according to the invention. The amino acid sequences are dividedinto all possible 13imers. The 13-mer peptides are sequentiallypresented to the models of the binding groove of the HLA-DR allotypesand a binding score assigned to each peptide for each allele. Aconformational score is calculated for each pocket-bound side chain ofthe peptide. This score is based on steric overlap, potential hydrogenbonds between peptide and residues in the binding groove, electrostaticinteractions and favorable contacts between peptide and pocket residues.The conformation of each side chain is then altered and the scorerecalculated. Having determined the highest conformational score, thebinding score is then calculated based on the (groove-bound hydrophobicresidues, the non-groove hydrophilic residues and the number of residuesthat fit into the binding groove. Known binders to MHC class II achievea significant binding score with almost no false negatives. Thuspeptides achieving, a significant binding score from the currentanalysis are considered to be potential T-cell epitopes. The results ofthe peptide threading analysis for the murine and veneered sequences areshown in Table 22.

Table 22. Potential T-cell epitopes in murine and veneered KS sequences.

TABLE 22 Potential T-cell epitopes in murine and veneered KS sequencesNumber of Location of potential potential T-cell epitopes (no. ofpotential Sequence epitopes MHC binders) Murine KS VH 6 35 (11), 62(17), 78 (12), 81 (12), 89 (6), 98 (15) Murine KS VH 5 30 (7), 62 (15),78 (11), 89 (6), 98 (15) Murine KS VK 6 1 (14), 2 (5), 17 (5), 27 (5),51 (13), 72 (18) Veneered KS VK 3 1 (17), 27 (5), 51 (13)5 Removal of Potential T Cell Epitopes

Potential T-cell epitopes are removed by making amino acid substitutionsin the particular peptide that constitutes the epitope. Substitutionswere made by inserting amino acids of similar physicochemical propertiesif possible. However in order to remove some potential epitopes, aminoacids of different size, charge or hydrophobicity may need to besubstituted. If changes have to made within CDRs which might have aneffect on binding, there is then a need to make a variant with andwithout the particular amino acid substitution. Numbering of amino acidresidues for substitution is as per Kabat. Potential T Cell epitopes arereferred to by the linear number of the first residue of the 13mer.

The amino acid changes required to remove T-cell epitopes from theveneered KS heavy chain variable region were as follows:

-   1. Substitution of arginine for lysine at residue 38 (Kabat    number 38) removes the potential epitope at residue no 30.-   2. Substitution of alanine for leucine at residue 72 (Kabat    number 71) and isoleucine for phenylalanine at residue 70 (Kabat    number 69) removes the potential epitope at residue 62. An    isoleucine at Kabat number 69 and alanine at Kabat number 71 is    found in a human germline VH sequence, DP10.-   3. Substitution of leucine for alanine at residue 79 (Kabat    number 78) removes the potential epitope at residue number 78.-   4. Substitution of threonine for methionine at residue 91 (Kabat    number 87), removes the potential epitope at residue number 89.-   5. Substitution of methionine for at isoleucine residue 100 (Kabat    number 96) in CDRH3 removes the potential epitope at residue 98.    There is no change out with CDRH3 which removes this potential    epitope.-   The amino acid substitutions required to remove the potential T-cell    epitopes from the veneered KS light chain variable region were as    follows:-   1. Substitution of isoleucine for methionine at residue 32 (Kabat    number 33) removes the potential epitope at residue number 27. This    residue is within CDR2. Isoleucine is commonly found at this    position in human antibodies.-   2. The potential epitope at position 1 is removed by substituting    valine for leucine at residue (Kabat number 3).-   3. Substitution of serine for alanine at residue 59 (Kabat    number 60) removes the potential epitope at residue number 51.    6 Design of De-immunized Sequences

De-immunized heavy and light chain sequences were designed withreference to the changes required to remove potential T-cell epitopesand consideration of framework residues that might be critical forantibody structure and binding. In addition to the de-immunizedsequences based on the veneered sequence, an additional sequence wasdesigned for each VH, and VK based on the murine sequence, termed theMouse Peptide Threaded (MoPT) version. For this version, changes weremade directly to the murine sequence in order to eliminate T-cellepitopes, but only changes outside the CDRs that are not considered tobe detrimental to binding are made. No attempt to remove surface (Bcell) epitopes has been made in this version of the de-immunizedsequence. The primary de-immunized VH includes substitutions 1 to 5 inSection 5 above and one extra change at residue 43 (Kabat number 43).Lysine found in the murine sequence was substituted for the glutaminefrom the human framework. Lysine is positively charged and thereforesignificantly different to glutamine; this region may be involved inVH/VL contacts. The primary de-immunized VH includes no potential T-cellepitopes. A further 4 de-immunized VHs were designed in order to testthe effect of the various substitutions required on antibody binding.The cumulative alterations made to the primary de-immunized sequence(KSDIVHv1) and the potential T-cell epitopes remaining are detailed inTable 23.

Table 23. Amino acid changes and potential epitopes in de-immunized KSVH.

TABLE 23 Amino acid changes and potential epitopes in de-immunized KS VHCumulative Potential epitopes residue (no. of potential MHC bindersVariant changes from 18 tested) KSDIVHv1 None none KSDIVHv2 96M → I 98(15) KSDIVHv3 71A → L, 78L → A 62 (16), 78 (11), 98 (15) KSDIVHv4 38 R →K 30 (7), 62 (16), 78 (11), 98 (15) KSDIVHv5 68T → A, 69I → F 30 (7), 62(17), 78 (11), 98 (15) KSMoPTVH NA 98 (15), 78 (12)

The primary de-immunized VK includes substitutions 1 to 3 in Section 5above. A further 3 de-immunized VKs were designed in order to test theeffect of the various substitutions required on antibody binding. Thecumulative alterations made to the primary de-immunized sequence(KSDIVKv1) and the potential T-cell epitopes remaining are detailed inTable 24. Sequences of modified epitopes of KS VH and KS VK are shown inTable 25.

Table 24. Amino acid changes and potential epitopes in de-immunized KSVK.

TABLE 24 Amino acid changes and potential epitopes in de-immunized KS VKPotential epitopes Cumulative residue (no. of potential MHC bindersVariant changes from 18 tested) KSDIVKv1 None none KSDIVKv2 33I → M 27(5) KSDIVKv3 3V → L 1 (17), 27 (5) KSDIVKv4 60 S → A 1 (17), 27 (5), 5(13) KSMoPTVK NA none

Table 25. Sequences of versions of modified epitopes of KS VH and KS VK.

TABLE 25 Sequences of versions of modified epitopes: KS VH veneered (SEQID NO: 658):QIQLVQSGPELKKPGSSVKISCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYADDFKGRFTFTLETSTSTAYLQLNNLRSEDMATYFCVRFISKGDYWGQGTTVTVSS KS VK veneered: (SEQ IDNO: 659):QILLTQSPASLAVSPGQRATITCSASSSVSYMLWYQQKPGQPPKPWIFDTSNLASGFPARFSGSGSGTSYTLTINSLEAEDAATYYCHQRSGYPYTFGGGTKVEIK KS de-immunized VH1 (SEQ ID NO:660):QIQLVQSGPELKKPGSSVKISCKASGYTFTNYGNNWVRQAPGKGLKWMGWINTYTGEPTYADDFKGRFTITAETSTSTLYLQLNNLRSEDTATYFCVRFMSKGDYWGQGTTVTVSS KS de-immunized VK1 (SEQID NO: 661):QIVLTQSPASLAVSPGQRATITCSASSSVSYILWYQQKPGQPPKPWIFDTSNLASGFPSRFSGSGSGTSYTLTINSLEAEDAATYYCHQRSGYPYTFGGGTKVEIK KS de-immunized VH2 (SEQ ID NO:662):QIQLVQSGPELKKPGSSVKISCKASGYTFTNYGMNWVRQAPGKGLKWMGWINTYTGEPTYADDFKGFTITAETSTSTLYLQLNNLRSEDTATYFCVRFISKGDYWGQGTTVTVSS KS de-immunized VK2 (SEQID NO: 663):QIVLTQSPASLAVSPGQRATITCSASSSVSYMLWYQQKPGQPPKPWIFDTSNLASGFPSRFSGSGSGTSYTLTINSLEAEDAATYYCHQRSGYPYTFGGGTKVEIK KS de-immunized VH3 (SEQ ID NO:664):QIQLVQSGPELKKPGSSVKISCKASGYTFTNYGMNNVRQAPGKGLKWMGWINTYTGEPTYADDFKGRFTITLETSTSTAYLQLNNLRSEDTATYFCVRFISKGDYWGQGTTVTVSS KS de-immunized VK3 (SEQID NO: 665):QILLTQSPASLAVSPGQRATITCSASSSVSYMLWYQQKPGQPPKPWIFDTSNLASGFPSRFSGSGSGTSYTLTINSLEAEDAATYYCHQRSGYPYTFGGGTKVEIK KS de-immunized VH4 (SEQ ID NO:666):QIQLVQSGPELKKPGSSVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFTITLETSTSTAYLQLNNLRSEDTATYFCVRFISKGDYWGQGTTVTVSS KS de-immunized VK4 (SEQID NO: 667):QILLTQSPASLAVSPGQRATITCSASSSVSYMLWYQQKPGQPPKPWIFDTSNLASGFPARFSCSGSGTSYTLTINSLEAEDAATYYCHQRSGYPYTFGGGTKVEIK KS de-immunized VH5 (SEQ ID NO:668):QIQLVQSGPELKKPGSSVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFTLETSTSTAYLQLNNLRSEDTATYFCVRFISKGDYWGQGTTVTVSS KS de-immunized VK5 (SEQID NO: 669):QILLTQSPASLAVSPGQRATITCSASSSVSYMLWYQQKPGSSPKPQIYDTSNLASGFPARFSGSGSGTSYTLTINSLEAEDAATYYCHQRSGYPYTFGGGTKVEIK KS VH mouse, peptide threaded (MoPT) (SEQ ID NO: 670):QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVRQAPGKGLKWMGWINTYTGEPTYADDFKGRFVFSLETSASTAFLQLNNLRSEDTATYFCVRFISKGDYWGQGTSVTVSS KS VK mouse, peptidethreaded (Mo PT) (SEQ ID NO: 671):QIVLTQSPATLSASPGERVTITCSASSSVSYMLWYLQKPGSSPKPWIFDTSNLASGFPSRFSGSGSGTTYSLIISSLEAEDAATYYCHQRSGYPYTFGGGTKLEIK KS VH mouse (SEQ ID NO: 672):QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQTPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAFLQINNLRNEDMATYFCVRFISKGDYWGQGTSVTVSS KS VK mouse (SEQ ID NO:673):QILLTQSPAIMSASPGEKVTMTCSASSSVSYMLWYQQKPGSSPKPWIFDTSNLASGFPARFSGSGSGTSYSLIISSMEAEDAATYYCHQRSGYPYTFGGGTKLEIKMAb 14.18-IL-2

In analogy, monoclonal antibody 14.18 was fused to IL-2 and deimmunizedaccording to the invention.

Potential T-cell epitopes in murine and veneered 14.18 sequences areshown in Table 26.

Table 26. Potential T-cell epitopes in murine and veneered MAb 14.18.

TABLE 26 Potential T-cell epitopes in murine and veneered MAb 14.18Number of potential Sequence T-cell Location of potential epitopesMurine 14.18 VH 11 3 (17), 9 (15), 30 (5), 35 (17), 39 (15), 43 (9), 58(12), 62 (11), 81 (11), 84 (16), 101 (7) Veneered 14.18 5 43 (9), 58(12), 62 (11), VH 81 (11), 84 (16) Murine 14.18 VK 7 7 (7), 13 (11), 27(15), 49 (11), 86 (17), 97 (11), 100 (4) Veneered 14.18 5 27 (15), 49(11), 86 (17), VK 97 (11), 100 (17)

Amino acid changes and potential epitopes in de-immunized 14.18 VH areshown in Table 27.

Table 27. Amino acid changes and potential epitopes in de-immunized14.18 VH.

TABLE 27 Amino acid changes and potential epitopes in de-immunized 14.18VH Potential epitopes Cumulative (no. of potential MHC Variant residuechanges binders from 18 tested) 14.18DIVH1 none none 14.18DIVH2 41I → P,45L → T, 50L → A none 14.18DIVH3 65S → G 58 (8) 14.18DIVH4 71A → V 58(8), 62 (4) 14.18DIVH5 45T → L, 41P → 1 43 (9) 58 (8) 62 (4) 14.18MoPTVHNA 43 (9) 58 (12) 62 (11)

Amino acid changes and potential epitopes in de-immunized 14.18 VK areshown in Table 28.

Table 28. Amino acid changes and potential epitopes in de-immunized14.18 VK.

TABLE 28 Amino acid changes and potential epitopes in de-immunized 14.18VH Potential epitopes' Cumulative (no. of potential MHC Variant residuechanges* binders from 18 tested) 14.18DIVKI None none 14.18DIVK2 46L →M, 49Y → H none 14.18DIVK3 96P → T, 100Q → G 97 (5) 14.18DIVK4 96T → L97 (11) 14.18DIVK5 27e S → R 27 (15), 97 (11) 14.18DIVK6 46M → L 27(15), 49 (11), 97 (11) 14.18MoPTVK NA 27 (15), 49 (11), 97 (11), 100 (4)

Sequences of versions of modified epitopes in de-immunized and veneered14.18 VH and VK are shown in Table 29.

Table 29. Sequences of versions of modified epitopes in de-immunized andveneered 14.18 VH and VK.

TABLE 29 Sequences of versions of modified epitopes in de-immunized andveneered 14.18 VH and VK 14.18 VH veneered (SEQ ID NO: 674):EVQLLQSGPELKKPGASVKISCKASGSSFTGYNNNWVRQAPGQRLEWIGAIDPYYGGTSYNQKFKGRATLSVDKSSSQAYMHLKSLTSEDSAVYYCVSGMEYWGQGTTVTVSS 14.18 VK veneered (SEQ IDNO: 675):DVVMTQSPGTLPVSLGERATISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLTISRLEAEDLAVYFCSQSTHVPPLTFGQGTKLEIK 14.18 de-immunized VH1 (SEQID NO: 676):EVQLLQSGPELKKPGASVKISCKASGSSFTGYNMNWVRQAIGQRLEWIGLIDPYYGGTSYNQKFKSRVTITADKSSSQAYMHLKSLTSEDTAVYYCVSGMEYWGQGTTVTVSS 14.18 de-immunized VK1 (SEQID NO: 677):DVVMTQSPGTLPVSLGERATISCRSSQSLVHSNGNTYLHWYLQKPGOSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRLEAEDMAVYFCSQSTHVPPPTFGQGTKVEIK 14.18 de-immunized VH2 (SEQID NO: 678):EVQLLQSGPELKKPGASVKISCKASGSSFTGYNMNWVRQAPGQRTEWIGAIDPYYGGTSYNQKFKSRVTITADKSSSQAYMHLKSLTSEDTAVYYCVSGMEYWGQGTTVTVSS 14.18 de-immunized VK2 (SEQID NO: 679):DVVMTQSPGTLPVSLGERATISCRSSQSLVHSNGNTYLHWYLQKPGQSPKMLIHKVSNRFSGVPDRFSGSGSGTDFTLTISRLEAEDMAVYFCSQSTHVPPPTFGQGTKVEIK 14.18 de-immunized VH3 (SEQID NO: 680):EVQLLQSGPELKKPGASVKISCKASGSSFTGYNMNWVRQAPGQRTEWIGAIDPYYGGTSYNQKFKGRVTITADKSSSQAYMHLKSLTSEDTAVYYCVSGMEYWGQGTTVTVSS 14.18 de-immunized VK3 (SEQID NO: 681):DVVMTQSPGTLPVSLGERATISCRSSQSLVHSNGNTYLHWYLQKPGQSPKMLIHKVSNRFSGVPDRFSGSGSGTDFTLTISRLEAEDMAVYFCSQSTHVPPTTFGGGTKVEIK 14.18 de-immunized VH4 (SEQID NO: 682):EVQLLQSGPELKKPGASVKISCKASGSSFTGYNMNWVRQAPGQRTEWIGAIDPYYGGTSYNQKFKGRVTITVDKSSSQAYMHLKSLTSEDTAVYYCVSGMEYWGQGTTVTVSS 14.18 de-immunized VK4 (SEQID NO: 683):DVVMTQSPGTLPVSLGERATISCRSSQSLVHSNGNTYLHWYLQKPGQSPKMLIHKVSNRFSGVPDRFSGSGSGTDFTLTISRLEAEDMAVYFCSQSTHVPPLTFGGGTKVEIK 14.18 de-immunized VH5 (SEQID NO: 684):EVQLLQSGPELKKPGASVKISCKASGSSFTGYNMNWVRQAIGQRLEWIGAIDPYYGGTSYNQKFKGRVTITVDKSSSQAYMHLKSLTSEDTAVYYCVSGMEYWGQGTTVTVSS 14.18 de-immunized VKS (SEQID NO: 685):DVVMTQSPGTLPVSLGERATISCRSSQSLVNGNTYLHWYLQKPGQSPKMLIHKVSNRFSGVPDRFSGSGSGTDFTLTISRLEAEDMAVYFCSQSTHVPPLTFGGGTKVEIK 14.18 VH mouse, peptidethreaded (Mo PT) (SEQ ID NO: 686):EVQLVQSGPEVEKPSASVKISCKASGSSFTGYNNNWVRQAIGKSLEWIGAIDPYYGGTSYMQKFKGRATLTVDKSSSTAYMHLKSLTSEDTAVYYCVSGMEYWGQGTTVTVSS 14.18 VK mouse, peptidethreaded (Mo PT) (SEQ ID NO: 687):DVVMTQTPGSLPVSAGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDSGVYFCSQSTHVPPLTFGAGTKLELK 14.18 VH mouse (SEQ ID NO:688):EVQLLQSGPELEKPSASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSS 14.18 VK mouse (SEQ ID NO:689):DVVMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK

The foregoing description and the examples are intended as illustrative,and are not to be taken as limiting. Still other variants within thespirit and scope of this invention are possible and will readily presentthemselves to those skilled in the art.

1. A modified fusion protein comprising an anti-KSA immunoglobulinmoiety and an IL-2 polypeptide moiety linked together directly orthrough a linker peptide; wherein the anti-KSA immunoglobulin moietycomprises an anti-KSA immunoglobulin variable region polypeptide whichcomprises an amino acid residue sequence selected from the groupconsisting of SEQ ID NO: 660, SEQ ID NO: 661, SEQ ID NO: 662, SEQ ID NO:663, SEQ ID NO: 664, SEQ ID NO: 665, SEQ ID NO: 666, SEQ ID NO: 667, SEQID NO: 668, SEQ ID NO: 669, SEQ ID NO: 676, SEQ ID NO: 677, SEQ ID NO:678, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQID NO: 683, SEQ ID NO: 684, and SEQ ID NO: 685.